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1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How long can pathogenic bacteria remain viable and airborne in a coughed aerosol?	4,286	for 45 min	44,465
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How far can pathogenic bacteria spread in a coughed aerosol?	4,287	4 m	44,513
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is considered the mechanism of human-to-human transmission of MERS-COV?	4,288	Droplet spread between humans	45,254
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the transmission of MERS-CoV is defined as?	4,289	sporadic	46,287
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the transmission of MERS-CoV is defined as?	4,290	intra-familial,	46,313
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the transmission of MERS-CoV is defined as?	4,291	often healthcare associated	46,329
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the transmission of MERS-CoV is defined as?	4,292	inefficient	46,358
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the transmission of MERS-CoV is defined as?	4,293	requiring close and prolonged contact	46,374
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the the rate of general transmission, even in outbreaks?	4,294	readily transmit to more than one other human	46,742
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What do the majority of human cases of MERS-CoV seem not to do?	4,296	readily transmit to more than one other human	46,742
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the basic reproduction number (R 0)?	4,298	the average number of infections caused by one infected individual in a fully susceptible population	46,950
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What is the basic reproduction number (R 0 ) for MERS-COV?	4,299	close to one	47,059
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	Why has MERS had a constant presence in the Arabian Peninsula?	4,300	due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks.	47,402
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What was the first known MERS human-to-human transmission event was one characterized by?	4,301	acute LRT disease	47,586
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	Where was the first known MERS human-to-human transmission event?	4,302	in a healthcare setting in Jordan	47,604
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	Approximately what percentage of MERS cases were fatal in KSA?	4,367	21 %	49,531
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	Approximately what percentage of MERS cases were died outside KSA?	4,368	21 %	49,531
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What percentage of HCWs comprised of MERS cases in the KSA and South Korea?	4,369	16 %	50,413
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How has most of the analysis of MERS-CoV genetics been performed?	4,370	using high throughput or "deep" sequencing methods for complete genome deduction	51,231
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What does Clade A contain?	4,372	only human-derived MERS-CoV genomes	52,035
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What does clade B comprise?	4,373	the majority of human and camel genomes deduced thus far	52,108
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How many clades have become apparent in genome of MERS-COV from humans and DCs?	4,371	has had more MERS cases than any other region of the KSA	58,320
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	Which city has had has had more MERS cases than any other region of the KSA ?	4,374	Riyadh	58,271
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	Which city harbours a wide range of MERS-CoV variants	4,375	Riyadh	58,270
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How has the vast majority of MERS-CoV transmission occurred?	4,376	from infected to uninfected humans in close and prolonged contact	66,077
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How were the transmission circumstances created?	4,377	by poor infection control in health care settings	66,173
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What percentage of humans have died among all humans reported to be infected?	4,378	nearly 40 %	67,310
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	What would aid accurate calculation of a case fatality ratio?	4,379	Global alignment of case definitions	67,480
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How does MARS-COV differ from SARS-COV?	4,380	MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.	68,137
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	Is there any evidence that MERS-CoV is a virus of pandemic concern?	4,381	There is no evidence that MERS-CoV is a virus of pandemic concern	69,043
1,741	MERS coronavirus: diagnostics, epidemiology and transmission https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687373/ SHA: f6fcf1a99cbd073c5821d1c4ffa3f2c6daf8ae29 Authors: Mackay, Ian M.; Arden, Katherine E. Date: 2015-12-22 DOI: 10.1186/s12985-015-0439-5 License: cc-by Abstract: The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases (ProMED) network on 20 th September 2012 [1] (Fig. 1) and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care [2] . The new virus was initially called novel coronavirus (nCoV) and subsequentlty entitled the Middle East respiratoy syndrome coronavirus (MERS-CoV). As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries (Additional file 1: Figure S1 ) confirmed by the World Health Organization (WHO), with over a third of the positive people dying (at least 527, 35 %) [3] . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA [4] , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA [5] . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure [10] . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26); that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS [14] ) [15] [16] [17] [18] [19] . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . The first WHO case definition [27] defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation [33] . Testing of asymptomatic people was recommended against from December 2014 [34] , reinforced by a case definition released by the KSA Ministry of Health in June 2015 [35] . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution [39, 40] . Among laboratory confirmed cases, fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia [37] . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . Disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . General supportive care is key to managing severe cases [43] . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % (n = 16) of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa [45] . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection [46] . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case [46] . Diagnostic methods were published within days of the ProMED email announcing the first MERS case [47] , including several now gold standard in-house RT-rtPCR assays (Fig. 2 ) as well as virus culture in Vero and LLC-MK2 cells [18, 47, 48] . A colorectal adenocarcinoma (Caco-2) epithelial cell line has since been recommended for isolation of infections MERS-CoV [49] . We previously [18] .). Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (UTR; grey rectangles). FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 [211] and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest RT-rtPCR screening assays and conventional, semi-nested (three primers) RT-PCR confirmatory sequencing assays [47, 48] . Publication order is noted by first [27 th September 2012; red] and second [6 th December 2012; orange] coloured rectangles; both from Corman et al. [47, 48] Those assays recommended by the WHO are highlighted underneath by yellow dots [53] . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] reviewed the broad tropism of MERS-CoV [5] . However, as is well described, cell culture is a slow, specialised and insensitive method [50] while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames (ORF 1a and 1b; Fig. 2 ) have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . The structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) [52] . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific [47, 48, 53] . The RealStar® kit uses these WHOrecommended assays [54] . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (IMM observation). Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . Additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR [61] . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity [62] . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA [63] . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV (see Molecular epidemiology: using genomes to understand outbreaks), serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype [49] . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction [64] . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . A number of commercial ELISA kits, immunofluorescent assays (IFA) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples [18, 48, 69] . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV [18] . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah (collected between Jan and Dec 2012) [70] . Of 226 slaughterhouse workers, only eight (3.5 %) were positive by IFA, and those sera could not be confirmed by virus neutralization (NT) test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA [70] . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive [71] . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool [31] . Since asymptomatic zoonoses have been posited [72] , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase [74, 75] . A pseudo particle neutralization (ppNT) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (MNT) test. [10, 74, [76] [77] [78] ] Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 [79, 80] . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay [10] . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. [46, 82, 83] This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein [71] . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A(H1N1) infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers [82] . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing [5, 84] . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray [84] is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) [69, 70, 85] or MNT test. [74, 85, 86] This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result [87] . The study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted [88, 89] . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . Recommended sample types include bronchoalveolar lavage (BAL), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . Fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°C [90] . If available, lung biopsy or autopsy tissues can also be tested [53] . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted [90] . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR [53, 90] . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more [25] . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable [83] . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred [2, 43, 93] . Higher viral loads occur in the LRT compared to the URT. [22, 69, 88, 94] This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease [21] . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR [95] . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . Despite this both the largest human MERS-CoV studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death [94] . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model [100] .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] In one instance, a HCW shed viral RNA for 42 days in the absence of disease [99] . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens [91, 102] . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (NPA; an URT sample), 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death [49, 94, 103] . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens [2, 25, 93, 104] . However, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . When viruses are sought, they have included human herpesvirus (HHV), rhinoviruses (HRV), enteroviruses (EV), respiratory syncytial virus (RSV), parainfluenzavirus types 1, 2 and 3 (PIVs),influenzaviruses (IFVs), endemic HCoVs, adenoviruses (AdVs) metapneumovirus (MPV) and influenza A\H1N1 virus; co-detections with MERS-CoV have been found on occasion [2, 22, 37, 69, 97] . Bacterial testing is sometimes included (for example, for Legionella and Pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses (negative for IFV, PIVs, RSV and AdVs) and RT-PCR for others (negative for AdV, EVs, MPV and HHVs) [18] . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested [47] . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. [110] A LRT sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 (hereafter called the Jeddah-2014 outbreak; Fig. 3 ), there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections (~3 % prevalence) [111] . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated HCWs (n = 1,695; 33.5 %) [32] . Among the detections, 19 (17.8 %) were HCWs and 10 (9.3 %) were family contacts [32] . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. [112] However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a "storm in a teacup". It is the low transmission rate that has prevented worldwide spread, despite many "opportunities". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of MERS-CoV with three of the first five cases having contact with DCs [73, 113, 114] . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease [115] . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs [116] . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC [117] . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles (Fig. 3) [118] . These factors may prove important for human cases who do not describe any DC contact [119] nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC [120] . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of "contact" during these interviews has been defined for one study [72] . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable (Fig. 4) [120] . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives [53, 125] . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA [121] . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported [85] and that link has matured into a verified association [38, 126] (Fig. 4) . (See figure on previous page.) Fig. 3 Monthly detections of MERS-CoV (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season [128] and when recently born DCs are weaned is indicated. Spring (green) and summer (orange) in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier [5] DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close [119] . Contact may be commonplace and could occur in variety of ways (Fig. 4a) . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA [127, 128] . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage [129] . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS [38] . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 [85] . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs (originally imported from the Horn of Africa) [85] . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island [5] . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody [85] . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates (UAE), Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands [85, [130] [131] [132] [133] [134] . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco (south American camelids) but none had detectable neutralising antibody against MERS-CoV [4, 74, 78, 85, 86, 135, 136] . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula [133] . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . From some of these, full or majority length genomes of MERS-CoV have been sequenced [77, 137, 138] . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 (as an antigenic facsimile for BCoV). It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans [117, 142, 143] . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative [76, 77, 144] . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . Viral loads among positive DCs can be very high [4, 76, 77, 139, 144] and DCs have been found positive both when ill with URT respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six (43 %) positive by two or more assays [138] . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences [138] . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant [138] . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners [117, 142, 146] . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre [142] . A rise in titre theoretically begins 10 to 21 days after DC infection [142] . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner [117, 142] . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection [142] . Camel calving season occurs in the winter months (between late October and late February; Fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations [128] . What role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections [129, 139, [147] [148] [149] . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h [150] . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity [150] . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar [151] . A single study has examined the ability of MERS-CoV to survive in the environment [150] . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity (RH) and virus recovery was attempted in cell culture. At high ambient temperature (30°C) and low RH (30 %) MERS-CoV remained viable for 24 h [150] . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions [150] . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % [150] . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV [152] . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . Whether MERS-CoV can remain adrift and infectious for extended periods (truly airborne) remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks [21, 23, 154, 155] . By extrapolation, aerosol-generating events involving DCs (urination, defecation, and preparation and consumption of DC products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] In a household study, 14 of 280 (5 %) contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining [157] [158] [159] [160] [161] . That is to say, the basic reproduction number (R 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 [162] , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards [162] . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . A rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases (549 of 1277) in the KSA were fatal betwen 2012 and December 2015 while 21 % (72 of 330) died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks (Additional file 2: Figure S2 ). Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections (Fig. 5) . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting [166] . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (IPC) procedures [115, 118] . Most of the analysis of MERS-CoV genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic [207] [208] [209] . Earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization [48] . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B (Fig. 6) . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far [168] . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today [77] . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene [170] and in the ORF1b region (Fig. 2) [172] . It is not unexpected that recombination should occur since it is well known among other CoVs [124] and because the majority of MERS-CoV whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences (downloaded from GenBank using the listed accession numbers and from virological.org [212] ). This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences (Geneious v8.1 [211] ). Clades are indicated next to dark (Clade A) or pale (Clade B) blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades [125, 168, 177] . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat [125] . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (Fig. 2) , which encodes the spike protein and accessory proteins [168] . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain (RBD) of the spike protein and codons 158 (N-terminal region), 460 (RBD), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants [172, 178] . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (Fig. 6 ) [169] . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact [175] . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants [128, 167, 179] . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them [73] . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection [162] . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint [182] and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive [25, 183] . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals [184] . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (Fig. 4c) . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male (68 M) travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 [187] . He developed fever, myalgia and a cough nearly a week later (11 th ). He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th [188] . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the South Korean outbreak. Approximately 34 people were infected during this time [187] . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died [189] . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China [91, 199, 200] . No contacts became ill. The outbreak was brought under control in late July/ early August [201] after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. [40] Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection [204] . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 [205] . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly [206] . Defining characteristics include the higher PFC among MERS cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS) and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The	How did the first WHO case definition define probable cases of MERS?	4,215	based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement.	6,732
2,527	On the Coronavirus (COVID-19) Outbreak and the Smart City Network: Universal Data Sharing Standards Coupled with Artificial Intelligence (AI) to Benefit Urban Health Monitoring and Management https://doi.org/10.3390/healthcare8010046 SHA: 90d04764b497a224a1d969f4e317fc19a5feab35 Authors: Allam, Zaheer; Jones, David S. Date: 2020 DOI: 10.3390/healthcare8010046 License: cc-by Abstract: As the Coronavirus (COVID-19) expands its impact from China, expanding its catchment into surrounding regions and other countries, increased national and international measures are being taken to contain the outbreak. The placing of entire cities in ‘lockdown’ directly affects urban economies on a multi-lateral level, including from social and economic standpoints. This is being emphasised as the outbreak gains ground in other countries, leading towards a global health emergency, and as global collaboration is sought in numerous quarters. However, while effective protocols in regard to the sharing of health data is emphasised, urban data, on the other hand, specifically relating to urban health and safe city concepts, is still viewed from a nationalist perspective as solely benefiting a nation’s economy and its economic and political influence. This perspective paper, written one month after detection and during the outbreak, surveys the virus outbreak from an urban standpoint and advances how smart city networks should work towards enhancing standardization protocols for increased data sharing in the event of outbreaks or disasters, leading to better global understanding and management of the same. Text: The novel Coronavirus outbreak, (previously known as the 2019-nCoV and later renamed COVID-19 during the writing of this manuscript) is leading to the closure of entire cities in China, and causing stringent measures to be taken in others. While in distant different continents, far from China where the virus was first reported, places are being placed on high alert. In Wuhan, where the virus broke, schools, roads and markets have been shut down [1] . The same is true in Hong Kong, Beijing and Hubei Province amongst surrounding areas, as precautionary measures are being emphasized to ensure that the spread of the virus is minimized, and complete and accurate information on the virus is being obtained [2] . However, the rate of spread of the virus and the uncertainties surrounding the entire situation has led the World Health Organization (WHO) on 30 January 2019 to declare the Coronavirus outbreak a 'Global Public Health Emergency'. WHO determined, however, not to declare the outbreak a 'Public Health Emergency of International Concern' (PHEIC) which is a higher level of declaration. A PHEIC is defined as "an extraordinary event which is determined to constitute a public health risk to other States through the international spread of disease and to potentially require a coordinated international response" whose scope may include: serious, sudden, unusual or unexpected; carries implications for public health beyond the affected State's national border; and may require immediate international action [3] . With the world having experienced some notable influenza pandemics in the past, a Global Initiative on Sharing All Influenza Data (GISAID) platform [4] was established and was instrumental in the rapid sharing of information by the Chinese scientists regarding the emergence of the COVID-19 virus. Through this platform, scientists from other regions were observed to gain access to information and are, subsequently, able to act in a much faster capacity; like in the case of scientists from the Virus Identification Laboratory based at Doherty Institute, Australia, who managed to grow a similar virus in the laboratory after accessing the data shared by the Chinese scientists [5] . Beyond the aspect of pandemic preparedness and response, the case of COVID-19 virus and its spread provide a fascinating case study for the thematics of urban health. Here, as technological tools and laboratories around the world share data and collectively work to devise tools and cures, similar efforts should be considered between smart city professionals on how collaborative strategies could allow for the maximization of public safety on such and similar scenarios. This is valid as smart cities host a rich array of technological products [6, 7] that can assist in early detection of outbreaks; either through thermal cameras or Internet of Things (IoT) sensors, and early discussions could render efforts towards better management of similar situations in case of future potential outbreaks, and to improve the health fabric of cities generally. While thermal cameras are not sufficient on their own for the detection of pandemics -like the case of the COVID-19, the integration of such products with artificial intelligence (AI) can provide added benefits. The fact that initial screenings of temperature is being pursued for the case of the COVID-19 at airports and in areas of mass convergence is a testament to its potential in an automated fashion. Kamel Boulos et al. [8] supports that data from various technological products can help enrich health databases, provide more accurate, efficient, comprehensive and real-time information on outbreaks and their dispersal, thus aiding in the provision of better urban fabric risk management decisions. The above improvements in the healthcare sector can only be achieved if different smart city products are fashioned to support standardized protocols that would allow for seamless communication between themselves. Weber and Podnar Žarko [9] suggest that IoT devices in use should support open protocols, and at the same time, the device provider should ensure that those fashioned uphold data integrity and safety during communication and transmission. Unfortunately, this has not been the case and, as Vermesan and Friess [10] explain, most smart city products use proprietary solutions that are only understood by the service providers. This situation often creates unnecessary fragmentation of information rendering only a partial integrated view on the dynamics of the urban realm. With restricted knowledge on emergent trends, urban managers cannot effectively take decisions to contain outbreaks and adequately act without compromising the social and economic integrity of their city. This paper, inspired by the case of the COVID-19 virus, explores how urban resilience can be further achieved, and outlines the importance of seeking standardization of communication across and between smart cities. With the advent of the digital age and the plethora of Internet of Things (IoT) devices it brings, there has been a substantial rise in the amount of data gathered by these devices in different sectors like transport, environment, entertainment, sport and health sectors, amongst others [11] . To put this into perspective, it is believed that by the end of 2020, over 2314 exabytes (1 exabyte = 1 billion gigabytes) of data will be generated globally [12] from the health sector. Stanford Medicine [12] acknowledges that this increase, especially in the medical field, is witnessing a proportional increase due to the increase in sources of data that are not limited to hospital records. Rather, the increase is being underpinned by drawing upon a myriad and increasing number of IoT smart devices, that are projected to exponentially increase the global healthcare market to a value of more than USD $543.3 billion by 2025 [13] . However, while the potential for the data market is understood, such issues like privacy of information, data protection and sharing, and obligatory requirements of healthcare management and monitoring, among others, are critical. Moreover, in the present case of the Coronavirus outbreak, this ought to be handled with care to avoid jeopardizing efforts already in place to combat the pandemic. On the foremost, since these cut across different countries, which are part of the global community and have their unique laws and regulations concerning issues mentioned above, it is paramount to observe them as per the dictate of their source country's laws and regulations; hence, underlining the importance of working towards not only the promoting of data through its usage but also the need for standardized and universally agreed protocols. While the significance of such data in advancing efficiency, productivity and processes in different sectors is being lauded, there are criticisms arising as to the nature of data collection, storage, management and accessibility by only a small group of users. The latter particularly includes select ICT corporations that are also located in specific geographies [6, [14] [15] [16] [17] . These criticisms are justified, as in recent years, big data is seen as the new 'gold rush' of the 21st century and limiting its access means higher economic returns and increased influence and control at various scales to those who control data. These associated benefits with big data are clearly influencing geopolitical standings, in both corporate and conventional governance realms, and there is increased competition between powerful economies to ensure that they have the maximum control of big data. As case in point is the amount of 'push and pull' that has arisen from Huawei's 5G internet planned rollout [18] . Though the latter service offers unprecedented opportunities to increase internet speeds, and thereby influence the handling of big data, countries like the U.S. and some European countries that are key proponents and players in global political, economic and health landscapes, are against this rollout, arguing that it is a deceptive way of gathering private data under the guise of espionage. On this, it has been noted that the issue of data control and handling by a few corporations accords with their principles of nationalism, and that these work for their own wellbeing as well as to benefit the territories they are registered in. Therefore, geopolitical issues are expected on the technological front as most large data-rich corporations are located in powerful countries that have influence both economically, health-wise and politically [19] [20] [21] . Such are deemed prized tokens on the international landscape, and it is expected that these economies will continue to work towards their predominant control as much as possible. On the health sector, the same approach is being upheld where critical information and data are not freely shared between economies as that would be seen to be benefiting other in-competition economies, whereas different economies would cherish the maximization of benefits from such data collections. In addition to the obvious deep-rooted social issues related to nationalism, other challenges include the increasing movement of people globally that is being enhanced by reduced costs and higher speed. In particular, these challenges are more pronounced when it comes to public health. This is because most of the health-related data collected not only can compromise local nations, but also captures those of travelers. In such cases, in a bid to improve the health status of a nation, it becomes paramount to factor in data from other regions necessitating unhindered sharing of this data. Such data-sharing truth is emphasized in situations like the recent case of Coronavirus outbreak threatening the global health environment, facilitated by air transportation. The virus was first reported in Wuhan, China, and in a matter of three weeks (by 17th January 2020) over 300 cases were confirmed in that region, and 10 days later (26th January 2020), a total of 2014 cases of Coronavirus have been reported, with 684 of those being confirmed, and with 29 reported outside China. The fatalities from the virus stands at 56 as of 26th January 2020 [22] . The virus had then been confirmed in various countries including Taiwan, South Korea, Japan, Thailand, France, the United States, Singapore and Vietnam [23] . In the above case, though major cities are known to prepare themselves for potential outbreaks, their health policies and protocols are observed to diverge from one another. Thus, without a global collaborative approach, progress towards working for a cure and universally acceptable policy approach can take longer. Such fears, of a lack of international collaboration, were highlighted by the World Health Organization (WHO) during an emergency meeting in Geneva on 22nd January 2020 to determine whether the virus outbreak had reached a level warranting international emergency concern. However, WHO was satisfied that China was being proactive in this case, unlike in 2002, when China withheld information on the outbreak for far too long, causing delays in addressing the epidemic [3] . As in this instance, it is the opinion in this paper that if there was seamless collaboration and seamless sharing of data between different cities, it would not warrant such a high-level meeting to result in action, and instead, a decision could have been made much earlier. On this, the saddest part is that some global cities are less prepared to handle the challenges posed by this type of outbreak for lack of information on issues like symptoms of the virus, the protective measures to be taken, and the treatment procedures that an infected person should be processed through, amongst other issues. The timely response by stakeholders in regard to this new outbreak are commendable compared to previous cases. The latter includes the Severe Acute Respiratory Syndrome (SARS) outbreak in 2002 that took substantial time (from November 2002 to April 2003) to identify and be dealt with [24] ; the Ebola outbreak in West Africa in 2013 that took months to determine; and the Zika Virus that was first reported in 2014 before being successfully identified in 2015. With the Coronavirus (COVID-19) , it took only 17 days (31st December 2019 to 17th January 2020) to be identified. The sharing of data has also been quicker, as immediately after the virus' genetic sequence was discovered, Chinese scientists were able to share the information with the WHO, thus helping in its identification and enabling the auctioning of precautionary measures in other countries. Latest technological tools have also allowed for the receipt of information in realtime, in contrast to traditional epidemiological approaches that would have required months to identify the outbreak type [25] . Similarly, though substantial data and information on the disease has been shared, Wetsman [26] acknowledges that there is a lack of some vital information, like the ease of spread of the virus from person-to-person, and this is a key to containing the disease as interactions between people from different parts of the globe are still active. This hindrance can be made further possible as many cities advance in their smart and safe city model implementation towards constructing sufficient soft and hard urban infrastructures equipped with, for example, thermal imagery sensors to allow for early detections. However, while that is the case, data access to many is a challenge because the information is often seen as being sensitive for national security reasons, whilst at the same time, acknowledging that a virus outbreak is an equal threat to both national security and the economy. The outbreak of any disease has significant impacts on local economies across the globe. For instance, when SARS (Severe Acute Respiratory Syndrome) (SARS-CoV) broke in China in 2002, it was estimated, that the Asian region incurred tremendous negative impacts socially, health-wise and economically, potentially amounting to Asian regional economy losses of between USD $12-18 billion from tourism, travel and retail sales industries alone [27] . The Zika virus outbreak, spread by daytime-active Aedes mosquitoes, is estimated to have cost equator-belt local economies in affected areas between USD $7 and USD $18 billion [28] . The Ebola virus (or Ebola hemorrhagic fever (EHF)) caused an estimated loss of USD $2.2 billion in GDP in three West African economies (Guinea, Liberia and Sierra Leone) in 2015 alone [29] . In regard to the current epidemic of Coronavirus, though it is too early to quantify or project its impacts on the global economy, there are fears that it may take the precedent of other outbreaks where billions of dollars will be lost. The foundations for this escalating loss can be witnessed in the rapid growth of travel bans being enacted by some countries and their international airports, especially specifically restricting people from visiting the affected regions in China and their growth into general non-Chinese travel movements. On this, noting that the outbreak came almost on the eve of the Lunar New Year celebrations, and that it had been estimated that over 400 million people were expected to travel in different parts of the world and China to observe this festivity, the majority have had to reconsider their options as to flights, hotels and entertainment events due to service provider cancellations [30] . Those who had already booked their flights are expected to receive their refunds following the directive by the Civil Aviation Administration of China, however, this move has already affected the share value of Chinese airline companies [30] . The above impacts demonstrate that the issues of virus outbreaks transcend urban safety and impacts upon all other facets of our urban fabric. Therefore, it becomes paramount to ensure that the measures taken to contain a virus transcend nationalist agendas where data and information sharing is normally restricted, to a more global agenda where humanity and global order are encouraged. With such an approach, it would be easier to share urban health data across geographies to better monitor emerging health threats in order to provide more economic stability, thereby ensuring no disruptions on such sectors like tourism and travel industries, amongst others. This is possible by ensuring collaborative, proactive measures to control outbreak spread and thus, human movements. This would remove fears on travelers, and would have positive impacts upon the tourism industry, that has been seen to bear the economic brunt whenever such outbreaks occur. This can be achieved by ensuring that protocols on data sharing are calibrated to remove all hurdles pertaining to sharing of information. On this, Lawpoolsri et al. [31] posits that such issues, like transparency, timelessness of sharing and access and quality of data, should be upheld so that continuous monitoring and assessment can be pursued. Virus outbreaks in recent years have shown that, in the urban realm, data, including health data, can be sourced from diverse places. Presently, in the case of Coronavirus (COVID-19) outbreak, data is being collected from airports through screening and monitoring, through the use of smart sensors installed in airport infrastructures and from personnel working in those air/seaports. For instance, it has been reported that in the U.S.A., screening is being carried out at 20 different airports to ensure that possible affected people are intercepted for quarantine at the point of entry. Beside airports, as reported by Buckley and May [2] , data is also being collected at bus terminals, market places (in Wuhan), subways, and also in health facilities where patients are taken for further medical attention. Such is prevalent especially in China, and other Asian regions where cases of the virus have been recorded and confirmed. In addition to these methods, other smart city data sources include the application of terminal tracking systems that are mostly emphasized in Safe City concepts, where, at the point of entry or departure, relevant data is collected and analyzed. Li et al. [32] highlights that sensors installed in such locations have the potential to receive and distribute data in real-time to digital infrastructures within the network, and their interconnectedness in the network renders them extremely efficient in providing real-time updates on different issues. Urban areas are also known to be amassed with numerous Urban Health sensors, some of which are wearable. Though these are not specifically fashioned to track the present case of virus outbreak, they are able to track other related parameters like heartbeat, blood pressure, body temperature and others variables, that when analyzed can offer valuable insights. Loncar-Turukalo et al. [33] hail these devices for their role in transforming the health care sector especially by allowing for Connected Health (CH) care, where data collected from them can be analyzed and provide insightful information on the health scenario in any given area. Vashist et al. [34] further highlight how emerging features such as spatiotemporal mapping, remote monitoring and management, and enhanced cloud computing capabilities can emanate from such endeavours, leading to better urban management potential. While it is true that the basic source of medical data is generally sourced from general practitioners or medical laboratories-a fact that has also been affirmed in the case of the current epidemic-this paper explores how data sourced from an urban perspective can contribute to the medical narrative. The conviction to dwell on the urban realm in this manuscript is based on the fact that the current epidemic (COVID-19) is transmitted majorly through human-to-human contact, and in most cases, especially where the spread is reported in a different country, the first point of contact is an urban area, where large groups of people convene, like airports or subway stations. In most cases, such facilities, which are mostly based in urban areas, are observed to have installed surveillance technologies to ensure that anyone showing any symptoms of the disease are identified and quarantined. However, even in such cases, as underlined in the present manuscript, the need for anonymizing medical data is emphasized to ensure that the use of current technologies does not breach data privacy and security requirements, across different geographies. In this case, novel technologies like Blockchain technologies and quantum cryptography can aid in the discussion and be made to integrate with data collecting technologies. This would render an increased wealth of data from both the medical field and smart city operators, while ensuring privacy and security; hence, aiding in providing relevant information for better informed decisions. However, despite the indisputable roles that installed devices play in providing relevant health information, their data communication aspect needs to be reviewed. First, communications are seen to be geography-restricted (restricted to a given location), such that they seldom expand or communicate with their like, installed beyond their restricted areas. Secondly, these devices are usually sourced and installed by separate corporations that maintain unique and specific standards for data processing and sharing, and accordingly, tying cities to the sole usage of their product(s). Such strategies are adopted as private corporations try to maximize their economic gains, since the digital solution market is a lucrative one and is expected to continue growing and expanding [6, 7] . For its current application, the standardization of protocols as elaborated in this manuscript need to be pursued to ensure that there is seamless sharing of information and data. By doing this, it is expected that issues like burdens of collecting data, accuracy and other complexity that are experienced (when systems are fragmented) are reduced or eliminated altogether. The standardization can be achieved by, for example, ensuring that all the devices and systems are linked into a single network, like was done in the U.S., where all the surveillance of healthcare were combined into the National Healthcare Safety Network (NHSH) [35] . The fact that cities are increasingly tuning on the concept of Smart Cities and boasting an increased adoption rate of technological and connected products, existing surveillance networks can be re-calibrated to make use of those new sets of databases. Appropriate protocols however have to be drafted to ensure effective actions while ensuring privacy and security of data and people. With scenarios like the present Coronavirus (COVID-19) outbreak, that not only impacts upon the economic status of cities, but also affects their social standing, it becomes imperative to emphasize the adoption of universal standards for data sharing. Such a move could have far reaching impact across cities and territories especially in positively combating outbreaks and disasters in a quicker, safer and standardized way, such that when the cure is discovered, the results can be replicated in various parts of the globe. With a collaborated data sharing protocol, it would be possible to have a larger dataset resulting in increased processing capabilities especially with technologies that are powered by artificial intelligence (AI) tools. Through this way, as noted by Jiang et al. [36] and Allam [37] , it would be possible to facilitate early detection, achieve better diagnosis and provide better urban management decisions for increased efficiency for virus containment. An example of how beneficial collaboration and sharing of data can be occurred during the 2014 Ebola outbreak in West Africa where scientists, health workers and clinicians, amongst other stakeholders from around the world, openly worked together and were able to contain the spread of this pandemic [38] . On this front, Boué et al. [39] highlight that levels of trust and transparency need to be reviewed and enhanced to facilitate unfettered data generation and sharing. Such could lead to an even earlier detection scenario of future virus outbreaks, and in the better curative management of the same, without minimal compromise on urban functions and on an urban economy. Furthermore, in cases of emergencies like the current outbreak of COVID-19 and any other, the need for observance of regulatory practices and international healthcare guidelines are paramount. This would ensure that both healthcare professionals and the general populace are informed, protected and remain within the prescribed rules and regulations. As noted by the WHO [40] , the healthcare guidelines and regulatory practices are advanced to also ensure that the health risk in question is reduced together with its consequences. In the current era of technological advancement, such regulations and guidelines are paramount as they have potential to lead to positive or negative outcomes. The position of this paper is to advance that it now possible to integrate technologies like the use of smart devices through IoT networks and wearable devices, data from mobile apps and others to help users to share information with accredited and certified health professionals, and in this case, improve the outcomes for better cross disciplinary and more resilient protocols and policies.	What is PHEIC?	1,238	Public Health Emergency of International Concern'	2,619
2,527	On the Coronavirus (COVID-19) Outbreak and the Smart City Network: Universal Data Sharing Standards Coupled with Artificial Intelligence (AI) to Benefit Urban Health Monitoring and Management https://doi.org/10.3390/healthcare8010046 SHA: 90d04764b497a224a1d969f4e317fc19a5feab35 Authors: Allam, Zaheer; Jones, David S. Date: 2020 DOI: 10.3390/healthcare8010046 License: cc-by Abstract: As the Coronavirus (COVID-19) expands its impact from China, expanding its catchment into surrounding regions and other countries, increased national and international measures are being taken to contain the outbreak. The placing of entire cities in ‘lockdown’ directly affects urban economies on a multi-lateral level, including from social and economic standpoints. This is being emphasised as the outbreak gains ground in other countries, leading towards a global health emergency, and as global collaboration is sought in numerous quarters. However, while effective protocols in regard to the sharing of health data is emphasised, urban data, on the other hand, specifically relating to urban health and safe city concepts, is still viewed from a nationalist perspective as solely benefiting a nation’s economy and its economic and political influence. This perspective paper, written one month after detection and during the outbreak, surveys the virus outbreak from an urban standpoint and advances how smart city networks should work towards enhancing standardization protocols for increased data sharing in the event of outbreaks or disasters, leading to better global understanding and management of the same. Text: The novel Coronavirus outbreak, (previously known as the 2019-nCoV and later renamed COVID-19 during the writing of this manuscript) is leading to the closure of entire cities in China, and causing stringent measures to be taken in others. While in distant different continents, far from China where the virus was first reported, places are being placed on high alert. In Wuhan, where the virus broke, schools, roads and markets have been shut down [1] . The same is true in Hong Kong, Beijing and Hubei Province amongst surrounding areas, as precautionary measures are being emphasized to ensure that the spread of the virus is minimized, and complete and accurate information on the virus is being obtained [2] . However, the rate of spread of the virus and the uncertainties surrounding the entire situation has led the World Health Organization (WHO) on 30 January 2019 to declare the Coronavirus outbreak a 'Global Public Health Emergency'. WHO determined, however, not to declare the outbreak a 'Public Health Emergency of International Concern' (PHEIC) which is a higher level of declaration. A PHEIC is defined as "an extraordinary event which is determined to constitute a public health risk to other States through the international spread of disease and to potentially require a coordinated international response" whose scope may include: serious, sudden, unusual or unexpected; carries implications for public health beyond the affected State's national border; and may require immediate international action [3] . With the world having experienced some notable influenza pandemics in the past, a Global Initiative on Sharing All Influenza Data (GISAID) platform [4] was established and was instrumental in the rapid sharing of information by the Chinese scientists regarding the emergence of the COVID-19 virus. Through this platform, scientists from other regions were observed to gain access to information and are, subsequently, able to act in a much faster capacity; like in the case of scientists from the Virus Identification Laboratory based at Doherty Institute, Australia, who managed to grow a similar virus in the laboratory after accessing the data shared by the Chinese scientists [5] . Beyond the aspect of pandemic preparedness and response, the case of COVID-19 virus and its spread provide a fascinating case study for the thematics of urban health. Here, as technological tools and laboratories around the world share data and collectively work to devise tools and cures, similar efforts should be considered between smart city professionals on how collaborative strategies could allow for the maximization of public safety on such and similar scenarios. This is valid as smart cities host a rich array of technological products [6, 7] that can assist in early detection of outbreaks; either through thermal cameras or Internet of Things (IoT) sensors, and early discussions could render efforts towards better management of similar situations in case of future potential outbreaks, and to improve the health fabric of cities generally. While thermal cameras are not sufficient on their own for the detection of pandemics -like the case of the COVID-19, the integration of such products with artificial intelligence (AI) can provide added benefits. The fact that initial screenings of temperature is being pursued for the case of the COVID-19 at airports and in areas of mass convergence is a testament to its potential in an automated fashion. Kamel Boulos et al. [8] supports that data from various technological products can help enrich health databases, provide more accurate, efficient, comprehensive and real-time information on outbreaks and their dispersal, thus aiding in the provision of better urban fabric risk management decisions. The above improvements in the healthcare sector can only be achieved if different smart city products are fashioned to support standardized protocols that would allow for seamless communication between themselves. Weber and Podnar Žarko [9] suggest that IoT devices in use should support open protocols, and at the same time, the device provider should ensure that those fashioned uphold data integrity and safety during communication and transmission. Unfortunately, this has not been the case and, as Vermesan and Friess [10] explain, most smart city products use proprietary solutions that are only understood by the service providers. This situation often creates unnecessary fragmentation of information rendering only a partial integrated view on the dynamics of the urban realm. With restricted knowledge on emergent trends, urban managers cannot effectively take decisions to contain outbreaks and adequately act without compromising the social and economic integrity of their city. This paper, inspired by the case of the COVID-19 virus, explores how urban resilience can be further achieved, and outlines the importance of seeking standardization of communication across and between smart cities. With the advent of the digital age and the plethora of Internet of Things (IoT) devices it brings, there has been a substantial rise in the amount of data gathered by these devices in different sectors like transport, environment, entertainment, sport and health sectors, amongst others [11] . To put this into perspective, it is believed that by the end of 2020, over 2314 exabytes (1 exabyte = 1 billion gigabytes) of data will be generated globally [12] from the health sector. Stanford Medicine [12] acknowledges that this increase, especially in the medical field, is witnessing a proportional increase due to the increase in sources of data that are not limited to hospital records. Rather, the increase is being underpinned by drawing upon a myriad and increasing number of IoT smart devices, that are projected to exponentially increase the global healthcare market to a value of more than USD $543.3 billion by 2025 [13] . However, while the potential for the data market is understood, such issues like privacy of information, data protection and sharing, and obligatory requirements of healthcare management and monitoring, among others, are critical. Moreover, in the present case of the Coronavirus outbreak, this ought to be handled with care to avoid jeopardizing efforts already in place to combat the pandemic. On the foremost, since these cut across different countries, which are part of the global community and have their unique laws and regulations concerning issues mentioned above, it is paramount to observe them as per the dictate of their source country's laws and regulations; hence, underlining the importance of working towards not only the promoting of data through its usage but also the need for standardized and universally agreed protocols. While the significance of such data in advancing efficiency, productivity and processes in different sectors is being lauded, there are criticisms arising as to the nature of data collection, storage, management and accessibility by only a small group of users. The latter particularly includes select ICT corporations that are also located in specific geographies [6, [14] [15] [16] [17] . These criticisms are justified, as in recent years, big data is seen as the new 'gold rush' of the 21st century and limiting its access means higher economic returns and increased influence and control at various scales to those who control data. These associated benefits with big data are clearly influencing geopolitical standings, in both corporate and conventional governance realms, and there is increased competition between powerful economies to ensure that they have the maximum control of big data. As case in point is the amount of 'push and pull' that has arisen from Huawei's 5G internet planned rollout [18] . Though the latter service offers unprecedented opportunities to increase internet speeds, and thereby influence the handling of big data, countries like the U.S. and some European countries that are key proponents and players in global political, economic and health landscapes, are against this rollout, arguing that it is a deceptive way of gathering private data under the guise of espionage. On this, it has been noted that the issue of data control and handling by a few corporations accords with their principles of nationalism, and that these work for their own wellbeing as well as to benefit the territories they are registered in. Therefore, geopolitical issues are expected on the technological front as most large data-rich corporations are located in powerful countries that have influence both economically, health-wise and politically [19] [20] [21] . Such are deemed prized tokens on the international landscape, and it is expected that these economies will continue to work towards their predominant control as much as possible. On the health sector, the same approach is being upheld where critical information and data are not freely shared between economies as that would be seen to be benefiting other in-competition economies, whereas different economies would cherish the maximization of benefits from such data collections. In addition to the obvious deep-rooted social issues related to nationalism, other challenges include the increasing movement of people globally that is being enhanced by reduced costs and higher speed. In particular, these challenges are more pronounced when it comes to public health. This is because most of the health-related data collected not only can compromise local nations, but also captures those of travelers. In such cases, in a bid to improve the health status of a nation, it becomes paramount to factor in data from other regions necessitating unhindered sharing of this data. Such data-sharing truth is emphasized in situations like the recent case of Coronavirus outbreak threatening the global health environment, facilitated by air transportation. The virus was first reported in Wuhan, China, and in a matter of three weeks (by 17th January 2020) over 300 cases were confirmed in that region, and 10 days later (26th January 2020), a total of 2014 cases of Coronavirus have been reported, with 684 of those being confirmed, and with 29 reported outside China. The fatalities from the virus stands at 56 as of 26th January 2020 [22] . The virus had then been confirmed in various countries including Taiwan, South Korea, Japan, Thailand, France, the United States, Singapore and Vietnam [23] . In the above case, though major cities are known to prepare themselves for potential outbreaks, their health policies and protocols are observed to diverge from one another. Thus, without a global collaborative approach, progress towards working for a cure and universally acceptable policy approach can take longer. Such fears, of a lack of international collaboration, were highlighted by the World Health Organization (WHO) during an emergency meeting in Geneva on 22nd January 2020 to determine whether the virus outbreak had reached a level warranting international emergency concern. However, WHO was satisfied that China was being proactive in this case, unlike in 2002, when China withheld information on the outbreak for far too long, causing delays in addressing the epidemic [3] . As in this instance, it is the opinion in this paper that if there was seamless collaboration and seamless sharing of data between different cities, it would not warrant such a high-level meeting to result in action, and instead, a decision could have been made much earlier. On this, the saddest part is that some global cities are less prepared to handle the challenges posed by this type of outbreak for lack of information on issues like symptoms of the virus, the protective measures to be taken, and the treatment procedures that an infected person should be processed through, amongst other issues. The timely response by stakeholders in regard to this new outbreak are commendable compared to previous cases. The latter includes the Severe Acute Respiratory Syndrome (SARS) outbreak in 2002 that took substantial time (from November 2002 to April 2003) to identify and be dealt with [24] ; the Ebola outbreak in West Africa in 2013 that took months to determine; and the Zika Virus that was first reported in 2014 before being successfully identified in 2015. With the Coronavirus (COVID-19) , it took only 17 days (31st December 2019 to 17th January 2020) to be identified. The sharing of data has also been quicker, as immediately after the virus' genetic sequence was discovered, Chinese scientists were able to share the information with the WHO, thus helping in its identification and enabling the auctioning of precautionary measures in other countries. Latest technological tools have also allowed for the receipt of information in realtime, in contrast to traditional epidemiological approaches that would have required months to identify the outbreak type [25] . Similarly, though substantial data and information on the disease has been shared, Wetsman [26] acknowledges that there is a lack of some vital information, like the ease of spread of the virus from person-to-person, and this is a key to containing the disease as interactions between people from different parts of the globe are still active. This hindrance can be made further possible as many cities advance in their smart and safe city model implementation towards constructing sufficient soft and hard urban infrastructures equipped with, for example, thermal imagery sensors to allow for early detections. However, while that is the case, data access to many is a challenge because the information is often seen as being sensitive for national security reasons, whilst at the same time, acknowledging that a virus outbreak is an equal threat to both national security and the economy. The outbreak of any disease has significant impacts on local economies across the globe. For instance, when SARS (Severe Acute Respiratory Syndrome) (SARS-CoV) broke in China in 2002, it was estimated, that the Asian region incurred tremendous negative impacts socially, health-wise and economically, potentially amounting to Asian regional economy losses of between USD $12-18 billion from tourism, travel and retail sales industries alone [27] . The Zika virus outbreak, spread by daytime-active Aedes mosquitoes, is estimated to have cost equator-belt local economies in affected areas between USD $7 and USD $18 billion [28] . The Ebola virus (or Ebola hemorrhagic fever (EHF)) caused an estimated loss of USD $2.2 billion in GDP in three West African economies (Guinea, Liberia and Sierra Leone) in 2015 alone [29] . In regard to the current epidemic of Coronavirus, though it is too early to quantify or project its impacts on the global economy, there are fears that it may take the precedent of other outbreaks where billions of dollars will be lost. The foundations for this escalating loss can be witnessed in the rapid growth of travel bans being enacted by some countries and their international airports, especially specifically restricting people from visiting the affected regions in China and their growth into general non-Chinese travel movements. On this, noting that the outbreak came almost on the eve of the Lunar New Year celebrations, and that it had been estimated that over 400 million people were expected to travel in different parts of the world and China to observe this festivity, the majority have had to reconsider their options as to flights, hotels and entertainment events due to service provider cancellations [30] . Those who had already booked their flights are expected to receive their refunds following the directive by the Civil Aviation Administration of China, however, this move has already affected the share value of Chinese airline companies [30] . The above impacts demonstrate that the issues of virus outbreaks transcend urban safety and impacts upon all other facets of our urban fabric. Therefore, it becomes paramount to ensure that the measures taken to contain a virus transcend nationalist agendas where data and information sharing is normally restricted, to a more global agenda where humanity and global order are encouraged. With such an approach, it would be easier to share urban health data across geographies to better monitor emerging health threats in order to provide more economic stability, thereby ensuring no disruptions on such sectors like tourism and travel industries, amongst others. This is possible by ensuring collaborative, proactive measures to control outbreak spread and thus, human movements. This would remove fears on travelers, and would have positive impacts upon the tourism industry, that has been seen to bear the economic brunt whenever such outbreaks occur. This can be achieved by ensuring that protocols on data sharing are calibrated to remove all hurdles pertaining to sharing of information. On this, Lawpoolsri et al. [31] posits that such issues, like transparency, timelessness of sharing and access and quality of data, should be upheld so that continuous monitoring and assessment can be pursued. Virus outbreaks in recent years have shown that, in the urban realm, data, including health data, can be sourced from diverse places. Presently, in the case of Coronavirus (COVID-19) outbreak, data is being collected from airports through screening and monitoring, through the use of smart sensors installed in airport infrastructures and from personnel working in those air/seaports. For instance, it has been reported that in the U.S.A., screening is being carried out at 20 different airports to ensure that possible affected people are intercepted for quarantine at the point of entry. Beside airports, as reported by Buckley and May [2] , data is also being collected at bus terminals, market places (in Wuhan), subways, and also in health facilities where patients are taken for further medical attention. Such is prevalent especially in China, and other Asian regions where cases of the virus have been recorded and confirmed. In addition to these methods, other smart city data sources include the application of terminal tracking systems that are mostly emphasized in Safe City concepts, where, at the point of entry or departure, relevant data is collected and analyzed. Li et al. [32] highlights that sensors installed in such locations have the potential to receive and distribute data in real-time to digital infrastructures within the network, and their interconnectedness in the network renders them extremely efficient in providing real-time updates on different issues. Urban areas are also known to be amassed with numerous Urban Health sensors, some of which are wearable. Though these are not specifically fashioned to track the present case of virus outbreak, they are able to track other related parameters like heartbeat, blood pressure, body temperature and others variables, that when analyzed can offer valuable insights. Loncar-Turukalo et al. [33] hail these devices for their role in transforming the health care sector especially by allowing for Connected Health (CH) care, where data collected from them can be analyzed and provide insightful information on the health scenario in any given area. Vashist et al. [34] further highlight how emerging features such as spatiotemporal mapping, remote monitoring and management, and enhanced cloud computing capabilities can emanate from such endeavours, leading to better urban management potential. While it is true that the basic source of medical data is generally sourced from general practitioners or medical laboratories-a fact that has also been affirmed in the case of the current epidemic-this paper explores how data sourced from an urban perspective can contribute to the medical narrative. The conviction to dwell on the urban realm in this manuscript is based on the fact that the current epidemic (COVID-19) is transmitted majorly through human-to-human contact, and in most cases, especially where the spread is reported in a different country, the first point of contact is an urban area, where large groups of people convene, like airports or subway stations. In most cases, such facilities, which are mostly based in urban areas, are observed to have installed surveillance technologies to ensure that anyone showing any symptoms of the disease are identified and quarantined. However, even in such cases, as underlined in the present manuscript, the need for anonymizing medical data is emphasized to ensure that the use of current technologies does not breach data privacy and security requirements, across different geographies. In this case, novel technologies like Blockchain technologies and quantum cryptography can aid in the discussion and be made to integrate with data collecting technologies. This would render an increased wealth of data from both the medical field and smart city operators, while ensuring privacy and security; hence, aiding in providing relevant information for better informed decisions. However, despite the indisputable roles that installed devices play in providing relevant health information, their data communication aspect needs to be reviewed. First, communications are seen to be geography-restricted (restricted to a given location), such that they seldom expand or communicate with their like, installed beyond their restricted areas. Secondly, these devices are usually sourced and installed by separate corporations that maintain unique and specific standards for data processing and sharing, and accordingly, tying cities to the sole usage of their product(s). Such strategies are adopted as private corporations try to maximize their economic gains, since the digital solution market is a lucrative one and is expected to continue growing and expanding [6, 7] . For its current application, the standardization of protocols as elaborated in this manuscript need to be pursued to ensure that there is seamless sharing of information and data. By doing this, it is expected that issues like burdens of collecting data, accuracy and other complexity that are experienced (when systems are fragmented) are reduced or eliminated altogether. The standardization can be achieved by, for example, ensuring that all the devices and systems are linked into a single network, like was done in the U.S., where all the surveillance of healthcare were combined into the National Healthcare Safety Network (NHSH) [35] . The fact that cities are increasingly tuning on the concept of Smart Cities and boasting an increased adoption rate of technological and connected products, existing surveillance networks can be re-calibrated to make use of those new sets of databases. Appropriate protocols however have to be drafted to ensure effective actions while ensuring privacy and security of data and people. With scenarios like the present Coronavirus (COVID-19) outbreak, that not only impacts upon the economic status of cities, but also affects their social standing, it becomes imperative to emphasize the adoption of universal standards for data sharing. Such a move could have far reaching impact across cities and territories especially in positively combating outbreaks and disasters in a quicker, safer and standardized way, such that when the cure is discovered, the results can be replicated in various parts of the globe. With a collaborated data sharing protocol, it would be possible to have a larger dataset resulting in increased processing capabilities especially with technologies that are powered by artificial intelligence (AI) tools. Through this way, as noted by Jiang et al. [36] and Allam [37] , it would be possible to facilitate early detection, achieve better diagnosis and provide better urban management decisions for increased efficiency for virus containment. An example of how beneficial collaboration and sharing of data can be occurred during the 2014 Ebola outbreak in West Africa where scientists, health workers and clinicians, amongst other stakeholders from around the world, openly worked together and were able to contain the spread of this pandemic [38] . On this front, Boué et al. [39] highlight that levels of trust and transparency need to be reviewed and enhanced to facilitate unfettered data generation and sharing. Such could lead to an even earlier detection scenario of future virus outbreaks, and in the better curative management of the same, without minimal compromise on urban functions and on an urban economy. Furthermore, in cases of emergencies like the current outbreak of COVID-19 and any other, the need for observance of regulatory practices and international healthcare guidelines are paramount. This would ensure that both healthcare professionals and the general populace are informed, protected and remain within the prescribed rules and regulations. As noted by the WHO [40] , the healthcare guidelines and regulatory practices are advanced to also ensure that the health risk in question is reduced together with its consequences. In the current era of technological advancement, such regulations and guidelines are paramount as they have potential to lead to positive or negative outcomes. The position of this paper is to advance that it now possible to integrate technologies like the use of smart devices through IoT networks and wearable devices, data from mobile apps and others to help users to share information with accredited and certified health professionals, and in this case, improve the outcomes for better cross disciplinary and more resilient protocols and policies.	How is PHEIC defined?	1,239	an extraordinary event which is determined to constitute a public health risk to other States through the international spread of disease and to potentially require a coordinated international response	2,740
2,527	On the Coronavirus (COVID-19) Outbreak and the Smart City Network: Universal Data Sharing Standards Coupled with Artificial Intelligence (AI) to Benefit Urban Health Monitoring and Management https://doi.org/10.3390/healthcare8010046 SHA: 90d04764b497a224a1d969f4e317fc19a5feab35 Authors: Allam, Zaheer; Jones, David S. Date: 2020 DOI: 10.3390/healthcare8010046 License: cc-by Abstract: As the Coronavirus (COVID-19) expands its impact from China, expanding its catchment into surrounding regions and other countries, increased national and international measures are being taken to contain the outbreak. The placing of entire cities in ‘lockdown’ directly affects urban economies on a multi-lateral level, including from social and economic standpoints. This is being emphasised as the outbreak gains ground in other countries, leading towards a global health emergency, and as global collaboration is sought in numerous quarters. However, while effective protocols in regard to the sharing of health data is emphasised, urban data, on the other hand, specifically relating to urban health and safe city concepts, is still viewed from a nationalist perspective as solely benefiting a nation’s economy and its economic and political influence. This perspective paper, written one month after detection and during the outbreak, surveys the virus outbreak from an urban standpoint and advances how smart city networks should work towards enhancing standardization protocols for increased data sharing in the event of outbreaks or disasters, leading to better global understanding and management of the same. Text: The novel Coronavirus outbreak, (previously known as the 2019-nCoV and later renamed COVID-19 during the writing of this manuscript) is leading to the closure of entire cities in China, and causing stringent measures to be taken in others. While in distant different continents, far from China where the virus was first reported, places are being placed on high alert. In Wuhan, where the virus broke, schools, roads and markets have been shut down [1] . The same is true in Hong Kong, Beijing and Hubei Province amongst surrounding areas, as precautionary measures are being emphasized to ensure that the spread of the virus is minimized, and complete and accurate information on the virus is being obtained [2] . However, the rate of spread of the virus and the uncertainties surrounding the entire situation has led the World Health Organization (WHO) on 30 January 2019 to declare the Coronavirus outbreak a 'Global Public Health Emergency'. WHO determined, however, not to declare the outbreak a 'Public Health Emergency of International Concern' (PHEIC) which is a higher level of declaration. A PHEIC is defined as "an extraordinary event which is determined to constitute a public health risk to other States through the international spread of disease and to potentially require a coordinated international response" whose scope may include: serious, sudden, unusual or unexpected; carries implications for public health beyond the affected State's national border; and may require immediate international action [3] . With the world having experienced some notable influenza pandemics in the past, a Global Initiative on Sharing All Influenza Data (GISAID) platform [4] was established and was instrumental in the rapid sharing of information by the Chinese scientists regarding the emergence of the COVID-19 virus. Through this platform, scientists from other regions were observed to gain access to information and are, subsequently, able to act in a much faster capacity; like in the case of scientists from the Virus Identification Laboratory based at Doherty Institute, Australia, who managed to grow a similar virus in the laboratory after accessing the data shared by the Chinese scientists [5] . Beyond the aspect of pandemic preparedness and response, the case of COVID-19 virus and its spread provide a fascinating case study for the thematics of urban health. Here, as technological tools and laboratories around the world share data and collectively work to devise tools and cures, similar efforts should be considered between smart city professionals on how collaborative strategies could allow for the maximization of public safety on such and similar scenarios. This is valid as smart cities host a rich array of technological products [6, 7] that can assist in early detection of outbreaks; either through thermal cameras or Internet of Things (IoT) sensors, and early discussions could render efforts towards better management of similar situations in case of future potential outbreaks, and to improve the health fabric of cities generally. While thermal cameras are not sufficient on their own for the detection of pandemics -like the case of the COVID-19, the integration of such products with artificial intelligence (AI) can provide added benefits. The fact that initial screenings of temperature is being pursued for the case of the COVID-19 at airports and in areas of mass convergence is a testament to its potential in an automated fashion. Kamel Boulos et al. [8] supports that data from various technological products can help enrich health databases, provide more accurate, efficient, comprehensive and real-time information on outbreaks and their dispersal, thus aiding in the provision of better urban fabric risk management decisions. The above improvements in the healthcare sector can only be achieved if different smart city products are fashioned to support standardized protocols that would allow for seamless communication between themselves. Weber and Podnar Žarko [9] suggest that IoT devices in use should support open protocols, and at the same time, the device provider should ensure that those fashioned uphold data integrity and safety during communication and transmission. Unfortunately, this has not been the case and, as Vermesan and Friess [10] explain, most smart city products use proprietary solutions that are only understood by the service providers. This situation often creates unnecessary fragmentation of information rendering only a partial integrated view on the dynamics of the urban realm. With restricted knowledge on emergent trends, urban managers cannot effectively take decisions to contain outbreaks and adequately act without compromising the social and economic integrity of their city. This paper, inspired by the case of the COVID-19 virus, explores how urban resilience can be further achieved, and outlines the importance of seeking standardization of communication across and between smart cities. With the advent of the digital age and the plethora of Internet of Things (IoT) devices it brings, there has been a substantial rise in the amount of data gathered by these devices in different sectors like transport, environment, entertainment, sport and health sectors, amongst others [11] . To put this into perspective, it is believed that by the end of 2020, over 2314 exabytes (1 exabyte = 1 billion gigabytes) of data will be generated globally [12] from the health sector. Stanford Medicine [12] acknowledges that this increase, especially in the medical field, is witnessing a proportional increase due to the increase in sources of data that are not limited to hospital records. Rather, the increase is being underpinned by drawing upon a myriad and increasing number of IoT smart devices, that are projected to exponentially increase the global healthcare market to a value of more than USD $543.3 billion by 2025 [13] . However, while the potential for the data market is understood, such issues like privacy of information, data protection and sharing, and obligatory requirements of healthcare management and monitoring, among others, are critical. Moreover, in the present case of the Coronavirus outbreak, this ought to be handled with care to avoid jeopardizing efforts already in place to combat the pandemic. On the foremost, since these cut across different countries, which are part of the global community and have their unique laws and regulations concerning issues mentioned above, it is paramount to observe them as per the dictate of their source country's laws and regulations; hence, underlining the importance of working towards not only the promoting of data through its usage but also the need for standardized and universally agreed protocols. While the significance of such data in advancing efficiency, productivity and processes in different sectors is being lauded, there are criticisms arising as to the nature of data collection, storage, management and accessibility by only a small group of users. The latter particularly includes select ICT corporations that are also located in specific geographies [6, [14] [15] [16] [17] . These criticisms are justified, as in recent years, big data is seen as the new 'gold rush' of the 21st century and limiting its access means higher economic returns and increased influence and control at various scales to those who control data. These associated benefits with big data are clearly influencing geopolitical standings, in both corporate and conventional governance realms, and there is increased competition between powerful economies to ensure that they have the maximum control of big data. As case in point is the amount of 'push and pull' that has arisen from Huawei's 5G internet planned rollout [18] . Though the latter service offers unprecedented opportunities to increase internet speeds, and thereby influence the handling of big data, countries like the U.S. and some European countries that are key proponents and players in global political, economic and health landscapes, are against this rollout, arguing that it is a deceptive way of gathering private data under the guise of espionage. On this, it has been noted that the issue of data control and handling by a few corporations accords with their principles of nationalism, and that these work for their own wellbeing as well as to benefit the territories they are registered in. Therefore, geopolitical issues are expected on the technological front as most large data-rich corporations are located in powerful countries that have influence both economically, health-wise and politically [19] [20] [21] . Such are deemed prized tokens on the international landscape, and it is expected that these economies will continue to work towards their predominant control as much as possible. On the health sector, the same approach is being upheld where critical information and data are not freely shared between economies as that would be seen to be benefiting other in-competition economies, whereas different economies would cherish the maximization of benefits from such data collections. In addition to the obvious deep-rooted social issues related to nationalism, other challenges include the increasing movement of people globally that is being enhanced by reduced costs and higher speed. In particular, these challenges are more pronounced when it comes to public health. This is because most of the health-related data collected not only can compromise local nations, but also captures those of travelers. In such cases, in a bid to improve the health status of a nation, it becomes paramount to factor in data from other regions necessitating unhindered sharing of this data. Such data-sharing truth is emphasized in situations like the recent case of Coronavirus outbreak threatening the global health environment, facilitated by air transportation. The virus was first reported in Wuhan, China, and in a matter of three weeks (by 17th January 2020) over 300 cases were confirmed in that region, and 10 days later (26th January 2020), a total of 2014 cases of Coronavirus have been reported, with 684 of those being confirmed, and with 29 reported outside China. The fatalities from the virus stands at 56 as of 26th January 2020 [22] . The virus had then been confirmed in various countries including Taiwan, South Korea, Japan, Thailand, France, the United States, Singapore and Vietnam [23] . In the above case, though major cities are known to prepare themselves for potential outbreaks, their health policies and protocols are observed to diverge from one another. Thus, without a global collaborative approach, progress towards working for a cure and universally acceptable policy approach can take longer. Such fears, of a lack of international collaboration, were highlighted by the World Health Organization (WHO) during an emergency meeting in Geneva on 22nd January 2020 to determine whether the virus outbreak had reached a level warranting international emergency concern. However, WHO was satisfied that China was being proactive in this case, unlike in 2002, when China withheld information on the outbreak for far too long, causing delays in addressing the epidemic [3] . As in this instance, it is the opinion in this paper that if there was seamless collaboration and seamless sharing of data between different cities, it would not warrant such a high-level meeting to result in action, and instead, a decision could have been made much earlier. On this, the saddest part is that some global cities are less prepared to handle the challenges posed by this type of outbreak for lack of information on issues like symptoms of the virus, the protective measures to be taken, and the treatment procedures that an infected person should be processed through, amongst other issues. The timely response by stakeholders in regard to this new outbreak are commendable compared to previous cases. The latter includes the Severe Acute Respiratory Syndrome (SARS) outbreak in 2002 that took substantial time (from November 2002 to April 2003) to identify and be dealt with [24] ; the Ebola outbreak in West Africa in 2013 that took months to determine; and the Zika Virus that was first reported in 2014 before being successfully identified in 2015. With the Coronavirus (COVID-19) , it took only 17 days (31st December 2019 to 17th January 2020) to be identified. The sharing of data has also been quicker, as immediately after the virus' genetic sequence was discovered, Chinese scientists were able to share the information with the WHO, thus helping in its identification and enabling the auctioning of precautionary measures in other countries. Latest technological tools have also allowed for the receipt of information in realtime, in contrast to traditional epidemiological approaches that would have required months to identify the outbreak type [25] . Similarly, though substantial data and information on the disease has been shared, Wetsman [26] acknowledges that there is a lack of some vital information, like the ease of spread of the virus from person-to-person, and this is a key to containing the disease as interactions between people from different parts of the globe are still active. This hindrance can be made further possible as many cities advance in their smart and safe city model implementation towards constructing sufficient soft and hard urban infrastructures equipped with, for example, thermal imagery sensors to allow for early detections. However, while that is the case, data access to many is a challenge because the information is often seen as being sensitive for national security reasons, whilst at the same time, acknowledging that a virus outbreak is an equal threat to both national security and the economy. The outbreak of any disease has significant impacts on local economies across the globe. For instance, when SARS (Severe Acute Respiratory Syndrome) (SARS-CoV) broke in China in 2002, it was estimated, that the Asian region incurred tremendous negative impacts socially, health-wise and economically, potentially amounting to Asian regional economy losses of between USD $12-18 billion from tourism, travel and retail sales industries alone [27] . The Zika virus outbreak, spread by daytime-active Aedes mosquitoes, is estimated to have cost equator-belt local economies in affected areas between USD $7 and USD $18 billion [28] . The Ebola virus (or Ebola hemorrhagic fever (EHF)) caused an estimated loss of USD $2.2 billion in GDP in three West African economies (Guinea, Liberia and Sierra Leone) in 2015 alone [29] . In regard to the current epidemic of Coronavirus, though it is too early to quantify or project its impacts on the global economy, there are fears that it may take the precedent of other outbreaks where billions of dollars will be lost. The foundations for this escalating loss can be witnessed in the rapid growth of travel bans being enacted by some countries and their international airports, especially specifically restricting people from visiting the affected regions in China and their growth into general non-Chinese travel movements. On this, noting that the outbreak came almost on the eve of the Lunar New Year celebrations, and that it had been estimated that over 400 million people were expected to travel in different parts of the world and China to observe this festivity, the majority have had to reconsider their options as to flights, hotels and entertainment events due to service provider cancellations [30] . Those who had already booked their flights are expected to receive their refunds following the directive by the Civil Aviation Administration of China, however, this move has already affected the share value of Chinese airline companies [30] . The above impacts demonstrate that the issues of virus outbreaks transcend urban safety and impacts upon all other facets of our urban fabric. Therefore, it becomes paramount to ensure that the measures taken to contain a virus transcend nationalist agendas where data and information sharing is normally restricted, to a more global agenda where humanity and global order are encouraged. With such an approach, it would be easier to share urban health data across geographies to better monitor emerging health threats in order to provide more economic stability, thereby ensuring no disruptions on such sectors like tourism and travel industries, amongst others. This is possible by ensuring collaborative, proactive measures to control outbreak spread and thus, human movements. This would remove fears on travelers, and would have positive impacts upon the tourism industry, that has been seen to bear the economic brunt whenever such outbreaks occur. This can be achieved by ensuring that protocols on data sharing are calibrated to remove all hurdles pertaining to sharing of information. On this, Lawpoolsri et al. [31] posits that such issues, like transparency, timelessness of sharing and access and quality of data, should be upheld so that continuous monitoring and assessment can be pursued. Virus outbreaks in recent years have shown that, in the urban realm, data, including health data, can be sourced from diverse places. Presently, in the case of Coronavirus (COVID-19) outbreak, data is being collected from airports through screening and monitoring, through the use of smart sensors installed in airport infrastructures and from personnel working in those air/seaports. For instance, it has been reported that in the U.S.A., screening is being carried out at 20 different airports to ensure that possible affected people are intercepted for quarantine at the point of entry. Beside airports, as reported by Buckley and May [2] , data is also being collected at bus terminals, market places (in Wuhan), subways, and also in health facilities where patients are taken for further medical attention. Such is prevalent especially in China, and other Asian regions where cases of the virus have been recorded and confirmed. In addition to these methods, other smart city data sources include the application of terminal tracking systems that are mostly emphasized in Safe City concepts, where, at the point of entry or departure, relevant data is collected and analyzed. Li et al. [32] highlights that sensors installed in such locations have the potential to receive and distribute data in real-time to digital infrastructures within the network, and their interconnectedness in the network renders them extremely efficient in providing real-time updates on different issues. Urban areas are also known to be amassed with numerous Urban Health sensors, some of which are wearable. Though these are not specifically fashioned to track the present case of virus outbreak, they are able to track other related parameters like heartbeat, blood pressure, body temperature and others variables, that when analyzed can offer valuable insights. Loncar-Turukalo et al. [33] hail these devices for their role in transforming the health care sector especially by allowing for Connected Health (CH) care, where data collected from them can be analyzed and provide insightful information on the health scenario in any given area. Vashist et al. [34] further highlight how emerging features such as spatiotemporal mapping, remote monitoring and management, and enhanced cloud computing capabilities can emanate from such endeavours, leading to better urban management potential. While it is true that the basic source of medical data is generally sourced from general practitioners or medical laboratories-a fact that has also been affirmed in the case of the current epidemic-this paper explores how data sourced from an urban perspective can contribute to the medical narrative. The conviction to dwell on the urban realm in this manuscript is based on the fact that the current epidemic (COVID-19) is transmitted majorly through human-to-human contact, and in most cases, especially where the spread is reported in a different country, the first point of contact is an urban area, where large groups of people convene, like airports or subway stations. In most cases, such facilities, which are mostly based in urban areas, are observed to have installed surveillance technologies to ensure that anyone showing any symptoms of the disease are identified and quarantined. However, even in such cases, as underlined in the present manuscript, the need for anonymizing medical data is emphasized to ensure that the use of current technologies does not breach data privacy and security requirements, across different geographies. In this case, novel technologies like Blockchain technologies and quantum cryptography can aid in the discussion and be made to integrate with data collecting technologies. This would render an increased wealth of data from both the medical field and smart city operators, while ensuring privacy and security; hence, aiding in providing relevant information for better informed decisions. However, despite the indisputable roles that installed devices play in providing relevant health information, their data communication aspect needs to be reviewed. First, communications are seen to be geography-restricted (restricted to a given location), such that they seldom expand or communicate with their like, installed beyond their restricted areas. Secondly, these devices are usually sourced and installed by separate corporations that maintain unique and specific standards for data processing and sharing, and accordingly, tying cities to the sole usage of their product(s). Such strategies are adopted as private corporations try to maximize their economic gains, since the digital solution market is a lucrative one and is expected to continue growing and expanding [6, 7] . For its current application, the standardization of protocols as elaborated in this manuscript need to be pursued to ensure that there is seamless sharing of information and data. By doing this, it is expected that issues like burdens of collecting data, accuracy and other complexity that are experienced (when systems are fragmented) are reduced or eliminated altogether. The standardization can be achieved by, for example, ensuring that all the devices and systems are linked into a single network, like was done in the U.S., where all the surveillance of healthcare were combined into the National Healthcare Safety Network (NHSH) [35] . The fact that cities are increasingly tuning on the concept of Smart Cities and boasting an increased adoption rate of technological and connected products, existing surveillance networks can be re-calibrated to make use of those new sets of databases. Appropriate protocols however have to be drafted to ensure effective actions while ensuring privacy and security of data and people. With scenarios like the present Coronavirus (COVID-19) outbreak, that not only impacts upon the economic status of cities, but also affects their social standing, it becomes imperative to emphasize the adoption of universal standards for data sharing. Such a move could have far reaching impact across cities and territories especially in positively combating outbreaks and disasters in a quicker, safer and standardized way, such that when the cure is discovered, the results can be replicated in various parts of the globe. With a collaborated data sharing protocol, it would be possible to have a larger dataset resulting in increased processing capabilities especially with technologies that are powered by artificial intelligence (AI) tools. Through this way, as noted by Jiang et al. [36] and Allam [37] , it would be possible to facilitate early detection, achieve better diagnosis and provide better urban management decisions for increased efficiency for virus containment. An example of how beneficial collaboration and sharing of data can be occurred during the 2014 Ebola outbreak in West Africa where scientists, health workers and clinicians, amongst other stakeholders from around the world, openly worked together and were able to contain the spread of this pandemic [38] . On this front, Boué et al. [39] highlight that levels of trust and transparency need to be reviewed and enhanced to facilitate unfettered data generation and sharing. Such could lead to an even earlier detection scenario of future virus outbreaks, and in the better curative management of the same, without minimal compromise on urban functions and on an urban economy. Furthermore, in cases of emergencies like the current outbreak of COVID-19 and any other, the need for observance of regulatory practices and international healthcare guidelines are paramount. This would ensure that both healthcare professionals and the general populace are informed, protected and remain within the prescribed rules and regulations. As noted by the WHO [40] , the healthcare guidelines and regulatory practices are advanced to also ensure that the health risk in question is reduced together with its consequences. In the current era of technological advancement, such regulations and guidelines are paramount as they have potential to lead to positive or negative outcomes. The position of this paper is to advance that it now possible to integrate technologies like the use of smart devices through IoT networks and wearable devices, data from mobile apps and others to help users to share information with accredited and certified health professionals, and in this case, improve the outcomes for better cross disciplinary and more resilient protocols and policies.	What platform was instrumental in rapid sharing of COVID-19 information?	1,240	Global Initiative on Sharing All Influenza Data (GISAID)	3,228
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What's the recommended procedure to disinfect at CT scanner after a COVID-19 exposure?	239	The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day.	18,441
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What's the recommended method to disinfect floors for COVID-19?	240	The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours.	19,312
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What is the role of computed tomography (CT) in COVID-19?	241	diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO	5,745
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What kind of masks are recommended to protect healthcare workers from COVID-19 exposure?	242	N95 mask	12,064
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What thickness of layers is recommended for CT image reconstruction in COVID-19 assessment?	243	1 mm-thick layers	15,491
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What must the data gathering include?	2,458	up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken.	22,205
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What must be done to assist in recognition of the disease on images and to allow accurate reporting of these findings?	2,459	All typical and atypical imaging features of the disease should be made known to all radiologists	22,369
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What is the major role of chest CT?	2,460	to understand the extent and dynamic evolution of lung lesions induced by COVID-19	24,384
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What is the reason to adopt low-dose CT?	2,461	low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients.	24,546
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What did the EMICT responsibilities include?	2,448	(1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally.	10,070
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	How were the radiology department areas divided?	2,449	contaminated, semicontaminated, buffer, and clean areas	10,860
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	How was the contaminated area connected to the CT room and other facilities?	2,450	connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents.	10,953
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What does the clean area include?	2,453	he clean area includes the administrative office and the diagnostic room.	11,603
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What does the semicontaminated area include?	2,451	the fever-CT control room, fever-DR control room, and other patient examination access areas.	11,408
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What does the buffer area include?	2,452	access areas for medical personnel and a dressing area for technologists.	11,528
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	How was the wearing and removing of the equipment performed?	2,454	under the supervision of the infection control nurse.	12,203
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What can lower the physical and mental stress levels of staff members?	2,455	periodically taking time off	12,348
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	Who must be assigned to the clean area?	2,456	Pregnant staff	12,636
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What responses must EMICT consider once a disease has been identified?	2,457	data gathering, collaboration, needs assessment, and expert advice	22,091
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What is the conclusion of the report?	2,462	Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control	4,062
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	What were the number of cases in mainland china as of March 11th?	2,463	80,793	4,738
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	How many COVID deaths occurred in Chinese mainland as of March 11th?	2,464	3,169	4,765
188	The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department https://www.jacr.org/article/S1546-1440(20)30285-4/pdf Journal Pre-proof Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song PII: S1546-1440(20)30285-4 DOI: https://doi.org/10.1016/j.jacr.2020.03.011 Reference: JACR 5139 To appear in: Journal of the American College of Radiology Received Date: 24 February 2020 Revised Date: 13 March 2020 Accepted Date: 15 March 2020 Please cite this article as: Huang Z, Zhao S, Li Z, Chen W, Zhao L, Deng L, Song B, The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department, Journal of the American College of Radiology (2020), doi: https://doi.org/10.1016/ j.jacr.2020.03.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of American College of Radiology The Battle Against Coronavirus Disease 2019 (COVID-19): Emergency Management and Infection Control in a Radiology Department Zixing Huang, Shuang Zhao, Zhenlin Li, Weixia Chen, Lihong Zhao, Lipeng Deng, Bin Song Department of Radiology, West China Hospital, Sichuan University, Chengdu, China *Zixing Huang and Shuang Zhao contributed equally to this work as co-first author. Corresponding Author: Bin Song, MD Address: Department of Radiology, West China Hospital, Sichuan University. No. 37, GUOXUE Alley, Chengdu, 610041, China Tel.: (+86)28 85423680, Fax: (+86)28 85582944 Email: [email protected]. Authors’ contributions ZXH: conceived the study and drafted the manuscript. ZS: conceived the study and drafted the manuscript. ZLL: The member of the emergency management and infection control team (EMICT) and was involved in the formulation of the measures. WXC: The member of the EMICT and was involved in the formulation of the measures. LHZ: The member of the EMICT and was involved in the formulation of the measures. LPD: The member of the EMICT and was involved in the formulation of the measures. BS: Leader of the EMICT, conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. The authors declare that they had full access to all of the data in this study and the authors take complete responsibility for the integrity of the data and the accuracy of the data analysis 1 The Battle Against Novel Coronavirus Pneumonia (COVID-19): Emergency Management and Infection Control in a Radiology Department Abstract Objective: To describe the strategy and the emergency management and infection control procedure of our radiology department during the COVID-19 outbreak. Methods: We set up emergency management and sensing control teams. The team formulated various measures: reconfiguration of the radiology department, personal protection and training of staff, examination procedures for patients suspected of or confirmed with COVID-19 as well as patients without an exposure history or symptoms. Those with suspected or confirmed COVID-19 infection were scanned in the designated fever-CT unit. Results: From January 21, 2020 to March 9, 2020, 3,083 people suspected of or confirmed with COVID-19 underwent fever-CT examinations. Including initial examinations and reexaminations, the total number of fever-CT examinations numbered 3,340. As a result of our precautions, none of the staff of the radiology department were infected with COVID-19. Conclusion: Strategic planning and adequate protections can help protect patients and staff against a highly infectious disease while maintaining function at a high volume capacity. Keywords: Coronavirus, COVID-19, novel coronavirus pneumonia, infection control 2 Introduction The whole world has been closely focusing on an outbreak of respiratory disease caused by a novel coronavirus that was first reported in Wuhan, China, on December 31, 2019, and that continues to spread. On February 11, 2020, the World Health Organization (WHO) named the disease “coronavirus disease 2019” (COVID-19). As of 24:00 on March 11, 2020, the National Health Commission (NHC) had received reports of 80,793 confirmed cases and 3,169 deaths on the Chinese mainland. There remain 14,831 confirmed cases (including 4,257 in serious condition) and 253 suspected cases still hospitalized. To date, 677,243 people have been identified as having had close contact with infected patients of whom13,701 are under medical observation [1]. Outside China, 44,067 laboratory-confirmed cases and 1,440 deaths have occurred in 117 countries /territories/areas according to the WHO [2]. COVID-19 poses significant threats to international health. Like the flu, COVID-19 is thought to spread mainly from person-to-person between people who are in close contact with one another through respiratory droplets produced when an infected person coughs or sneezes. In light of the infectious nature of this disease, healthcare workers are at high risk of infection of COVID-19. In China, healthcare workers account for 1,716 confirmed cases of COVID-19, including six deaths [3]. Computed tomography (CT) can play a role in both diagnosing and categorizing COVID-19 on the basis of case definitions issued by the WHO and the treatment guidelines from the NHC [4]. Suspected patients having the virus may undergo chest CT. Isolation and barrier procedures are necessary to protect both the department staff and other patients in the hospital. Note should be made that due to overlap of imaging findings with other respiratory 3 diseases, CT is not helpful as a screening tool. But it can help identify the degree of pulmonary involvement and disease course. Our hospital is a national regional medical center with 4,300 beds and a tertiary referral center in Sichuan province. The initial response started on January 21, 2020, after transmission of COVID-19 was confirmed to be human-to-human on January 20, 2020. The first suspected case of COVID-19 in Sichuan province was reported on January 21, 2020. The Sichuan provincial government immediately launched the first-level response to major public health emergencies. On the same day, our hospital was designated to care for Sichuan province patients with COVID-19. This article describes the emergency management procedure of our radiology department for situations involving severe infectious diseases, such as COVID-19, and the infection-protection experience of the department staff. Methods The hospital provided personal protective equipment (medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles) to all its healthcare staff, erected three medical tents (fever tents) for screening of fever cases in the parking lot of the emergency department, planned an examination route and examination area for patients suspected of harboring the virus, and placed confirmed patients in an isolation ward. “Fever” was the colloquial term used to designate suspected COVID-19 based on symptoms such as a fever or with an epidemiological history of a potential exposure as well as those with confirmed COVID-19 referred for treatment. Further, during outbreak, emergency and outpatient patients 4 without fever were asked for information such as epidemiological history and sent to fever tents as long as they met suspected criteria. The radiology department has 65 diagnostic radiologists and 161 other staff members (trained technologists, nurses, engineers, and support staff). The equipment of the radiology department includes 12 magnetic resonance (MR) scanners, 14 CT scanners, 15 digital subtraction angiography (DSA) systems, 32 sets of digital radiography (DR) systems (including nine mobile bedside DR sets), and 130 imaging diagnostic workstations for picture archiving and communication systems (PACS). Most of the equipment is distributed among four buildings at the hospital main campus. 4 CT scanners, 4 MR scanners, 1 DR are located on the first floor of the first inpatient building, and 9 DR and 8 DSA are located on the second floor. 1 CT and 1 MR scanner are located in the third inpatient building. 1 CT and 1 MR scanner are located in the sixth inpatient building. 2 CT scanners, 2 MR scanners and 7 DSA are located in the technical building. The rest of the equipment is located in the seventh inpatient building in the branch campus. The first inpatient building, located next to the emergency department, was reconfigured to handle cases of COVID-19. Fever tents were set up by the emergency department in the emergency department parking lot to separate normal emergency patients from patients with symptoms or exposure history suspicious of COVID-19. We established separate means of access between fever tents and between the fever examination area of the radiology department to avoid cross-contamination. The emergency management and infection control measures, as described below and implemented in the radiology department during the outbreak, have been approved by the 5 infection control committee of hospital. These measures are in accordance with relevant laws and regulations, in order to protect patients as well as the staff. Radiology Emergency Management and Infection Control Team (EMICT) The radiology department director chaired the EMICT. Its members include the deputy director, chief technologist, head nurse, equipment engineer supervisor, and infection control nurse of the radiology department. Team responsibilities included (1) coordination between the hospital’s management and planning of infection control and radiology departments; (2) collection of the most up-to-date protection-related information to educate and train staff in the department; (3) reallocation of staff according to the actual situation; (4) establishment of the CT procedures for patients with COVID-19; and (5) establishment of an emergency management plan for the radiology department to ensure that the department would run normally. Suspected patients The suspected patients were identified according to the Diagnosis and Treatment Program of the Novel Coronavirus Pneumonia of the NHC [5], mainly based on epidemiological history. Reconfiguration of the radiology department The radiology department was divided into four areas [6]: contaminated, semicontaminated, buffer, and clean areas (Figure 1). The contaminated area is connected to the fever clinic and includes the fever accessway, the CT examination room, and the DR examination room for 6 confirmed and suspected cases. One CT scanner and one DR system closest to the emergency department are designated the fever-CT and fever-DR to examine patients with suspected and confirmed COVID-19. There is a separate dedicated access between the contaminated area and the fever screening tents. The semicontaminated area includes the fever-CT control room, fever-DR control room, and other patient examination access areas. The buffer zone includes access areas for medical personnel and a dressing area for technologists. The clean area includes the administrative office and the diagnostic room. The contaminated area was isolated from other areas using physical barricades. Directional signs were newly installed to guide patients and staff. Personal protection and training of staff For providing care for patients with confirmed and suspected COVID-19, all hospital staff are required to wear complete personal protective equipment [7]: medical protective clothing, surgical cap, N95 mask, gloves, face shields, and goggles. Wearing and removing of the equipment must be performed in accordance with the procedures and under the supervision of the infection control nurse. Because staff members working in the contaminated area are under much situational pressure, periodically taking time off could lower their physical and mental stress levels. The technologists on fever-CT duty shifts are provided a break once a week for four hours. In addition, the health of staff in the contaminated area must be monitored closely for the symptoms of COVID-19. Pregnant staff must be assigned to the clean area. 7 The EMICT formulates and continually updates guidelines and educates all staff for West China Hospital of Sichuan University. The EMICT training for staff is mainly involves documents regarding infection control and CT findings of COVID-19 and maintains an EMICT WeChat group for West China Hospital of Sichuan University. WeChat is the most widely used social media app in China. The EMICT releases the latest national and hospital-based information regarding COVID-19, guidance documents, and other notices from the hospital and radiology department in the WeChat group on a daily basis. Staff can also report to the EMICT in the WeChat group any time. Protocols for each modality and infection control instructions are posted on the walls in all examination rooms. The EMICT periodically reminds staff to undertake personal measures to reduce infection, such as wearing masks at all instances in the radiology department and N95 masks if working in the contaminated area; not touching the mask and the eyes; practicing hand hygiene; facing away from colleagues when eating, drinking, and talking; and not using personal cell phones while on duty. In addition, the chief thoracic radiologist provided lectures on all radiologists and technologists on typical CT findings of COVID-19 infection using materials developed in Wuhan, the epicenter of the outbreak in China. CT examination procedures There are two sets of procedures for CT examination: the fever-CT procedure and routine CT procedure for those not suspected of COVID-19. The fever-CT procedure for suspected or confirmed COVID-19 (Figure 2) 8 Before the fever-CT technologist operates the equipment, he or she should wear personal protective equipment according to three-level protection standard [8]. Before the CT examination of patients with suspected and confirmed COVID-19 begins, the fever tent or isolation ward notifies the radiologist in advance. The fever-CT technologist checks the equipment and prepares to disinfect the imaging equipment immediately after the examination. The patient enters the fever-CT waiting area through the fever access area. If the patient can get onto and off the examination table by themselves, the patient is allowed to do so. If the patient cannot get onto or off the examination table independently, the person accompanying the patient assists the patient, rather than the technologist. The technologist checks the patient information and, using an intercom system in the examination room, asks the patient to remove any metal ornaments on the neck and chest. Also, by intercom, the technologist trains the patient to hold his or her breath during the examination. The technologist uses a low-dose chest CT protocol to scan the patient. After scanning, the original images are reconstructed as 1 mm-thick layers. The technologist browses the images to ensure that their quality meets the diagnostic requirements and then guides the patient to leave through the fever access area. The disposable sheets for patient examination are changed after each patient. The equipment is disinfected according to the procedure below. To protect themselves, the technologists assigned to the fever-CT wear N95 mask and other personal protection as established by the EMICT. The CT procedure for regular patients (figure.3) 9 Some patients with COVID-19 have no symptoms, and they may call at the general clinic for other reasons. The following CT procedure is applicable under these circumstances: When the patient makes an appointment for examination, the staff asks the patient about their epidemiological history, symptoms, and signs. If suspected criteria are met, the patient will be sent to the fever tent for further screening. When a patient presents to the radiology department entrance, his/her temperature is measured. If the temperature is higher than 37.2 , ℃ the patient is sent to the fever tent for further investigation. Those with no exposure history, suspicious symptoms or fever are screened in one of the non-contaminated CT scanners. The technologists assigned to these scanners wear surgical masks. All patients and the person accompanying them are required to wear surgical masks. After the CT examination, the technologist browses the images quickly. If the CT appearance is typical of lung infection, the technologist immediately reports it to the chest radiologist on duty and asks the patient to wait in the CT examination room. If the chest radiologist does not suspect COVID-19 infection, the patient can leave the CT examination room. If the chest radiologist does suspect COVID-19 infection, the technologist immediately reports it to the EMICT and sends the patient to the fever tent. The floor and equipment in the CT examination room are disinfected according to regulations, and air disinfection is conducted for 30 min before examining other patients. These CT scanners are considered noncontaminated (not fever-CTs) after these sterilization procedures. Fever-DR examination procedure 10 The COVID-19 guideline of the NHC does not recommend chest DR because its ability in diagnosing COVID-19 is limited. At our hospital, we only use mobile DR units to provide bedside examination for critically ill patients. The technologist operating the mobile DR wears personal protective equipment according to the three-level protection standard and sterilizes the mobile DR according to the ward management requirements as described below. Equipment and environment disinfection procedures Routine disinfection procedure [9] 1) Object surface disinfection: Object surface is wiped with 1000mg/L chlorine-containing disinfectant, wipe twice with 75% ethanol for non-corrosion resistance, once /4 hours. 2) Equipment disinfection: The equipment in the contaminated area are wiped with 2000mg/L chlorine-containing disinfectant. The DR and CT gantry in the contaminated area are wiped with 75% ethanol. The equipment in the buffer area is wiped with 500-1000mg/L chlorine-containing disinfectant or alcohol-containing disposable disinfectant wipes twice a day. 3) Air disinfection: Turning off all central air conditioners to prevent air contamination with each other. Polluted area: open the door for ventilation, each time more than 30 minutes, once /4 hours; The air sterilizer is continuously sterilized or the ultraviolet ray is continuously used in the unmanned state for 60 minutes, four times a day, remembered to close the inner shielding door when air disinfection. Other ambient air is sprayed with 1000mg/L chlorine-containing disinfectant and ventilated twice a day 4) Ground disinfection: The ground is wiped with 1000mg/L chlorine-containing disinfectant, once /4 hours. 5) When contaminated, disinfect at any time. In case of visible contamination, disposable absorbent materials should be used first to completely remove the pollutants, and then a cloth soaked with 2000mg/L chlorine-containing disinfectant should be used for 30 minutes before wiping. 11 Fever-CT disinfection procedures after examination In addition to the above, disinfect the examination bed and ground with chlorinated disinfectant containing 2000mg/L [10]. Noncontaminated CT disinfection procedures after suspected COVID-19 case examination In addition to the above routine disinfection procedure, air disinfection is conducted for 30 min before examining other patients. Results From January 21, 2020 when screening for epidemiological history or symptoms suspicious for COVID-19, to March 9, 2020, our hospital screened a total of 7,203 individuals and confirmed 24 cases of COVID-19. Of these, 3,083 people underwent fever-CT examinations. Including the initial examination and reexamination, the total number of fever CT examination numbered 3,340. The fever-CT scanned a patient approximately every 21.5 minutes. As a result of our precautions, none of the staff of the radiology department developed symptoms suspicious for COVID-19. The fever-CT technologist, with the highest probability of exposure, remains PCR negative. Discussion It has been 17 years since the severe acute respiratory syndrome (SARS) epidemic, the last national spread of severe infectious disease, broke out. Currently, the Chinese people are panicking again. The speed and extent by which COVID-19 has spread in 2 months are 12 unprecedented, beyond those of SARS, and this has been aided by its contagious nature and rapid spread via droplets and contact. The droplet mode of transmission means that a person can be infected easily by means of casual contact or even fomites on contaminated environmental surfaces. Another theory has yet to be proved: aerosol propagation. How radiology departments respond to any infectious disease outbreak is determined primarily by the estimated risk of cross-infection to the staff and other patients. Appropriate precautions taken only by staff in direct contact with patients may be adequate when the risk is low. The strongest measures need to be implemented to limit the spread of the disease when the risk is high. With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented; these include providing adequate standard protective equipment, training staff, and instituting proper emergency plans. Once a contagious infectious disease has been identified, the EMICT must consider four main areas of response: data gathering, collaboration, needs assessment, and expert advice [10]. Data gathering includes dissemination of up-to-date case definitions and information about confirmatory tests to all staff with direct patient contact to allow appropriate barrier precautions to be taken. All typical and atypical imaging features of the disease should be made known to all radiologists to assist in recognition of the disease on images and to allow accurate reporting of these findings. We have stored images of all probable cases of COVID-19 in the PACS so that these images were readily available for any radiologist to review, and images from previous imaging studies are also available for comparison. Collaboration with the radiology departments of other hospitals is very important because patients may initially present to different centers, depending on geographic location and travel 13 distance. These patients may be few in number at a single hospital, but if data from patients at several hospitals are available, a more accurate overall understanding of both imaging features and epidemiology can be achieved. Dissemination of this information to all healthcare facilities will also lead to early recognition of the disease, and appropriate isolation measures may be instituted. The Internet and social media apps, especially WeChat, have been used for distribution of medical information, and because the exchange of information regarding infectious disease outbreaks is almost instantaneous, it is an indispensable tool for radiologists. In fact, within a month of the outbreak, the hospital that received the most infected patients from the source of the outbreak made a PowerPoint presentation of the CT manifestations of COVID-19, which was shared via WeChat and disseminated across the country in a very short time. Subsequently, COVID-19-teaching PowerPoint presentations from various hospitals appeared and were quickly shared via WeChat. Our diagnostic process is limited as chest CT along is not diagnostic of COVID-19 because of lack of imaging specificity. But when combined with other epidemiological, clinical, laboratory and virus nucleic acid information, typical chest CT imaging findings are helpful for making the diagnosis. In our opinion, the major role of chest CT is to understand the extent and dynamic evolution of lung lesions induced by COVID-19. The reasons why we adopted the low-dose chest CT scan protocol are as follows: low-dose chest CT has been widely used in the screening of early lung cancer. It is well known that many early lung cancers are ground-glass opacities (GGO), so we believe that low-dose screening is also applicable for COVID-19. In addition, considering the rapid development of COVID-19, many CT 14 examinations may be conducted in the same individual to monitor disease progress. Low-dose scanning can reduce the radiation damage to patients. Although the processes we established minimized the exposure of hospital staff, ancillary personnel and other patients, it remains limited as follows. Sichuan province is not the center of the epidemic. The number of patients with COVID-19 whom we have treated has not been high, and most cases are from other provinces of China. However, we believe that our experience in management, the reconfiguration of our radiology department, and the workflow changes implemented in the current COVID-19 situation are useful for other radiology departments that must prepare for dealing with patients with COVID-19. While no radiology personnel developed symptoms suspicious for or were confirmed as having COVID-19, there may be asymptomatic personnel. REFERENCES 1. National Health Commission of the People’s Republic of China.(2020). March 12: Daily briefing on novel coronavirus cases in China. Retrieved from http://en.nhc.gov.cn/2020-03/12/c_77618.htm. Accessed March 11, 2020. 2. World Health Organization. (2020). Coronavirus disease 2019 (COVID-19) Situation Report-52. Retrieved from https://www.who.int/docs/default-source/coronaviruse/20200312-sitrep-52-covid-19.pdf?sfvrsn=e 2bfc9c0_2 9. Accessed March 11, 2020. 3. National Health Commission of the People’s Republic of China.(2020). Latest developments in epidemic control on Feb 15. Retrieved from http://en.nhc.gov.cn/2020-02/16/c_76622. Accessed March 11, 2020. 15 4. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020 5. Health Commission of the People’s Republic of China.(2020). The notification of the trial operation based on the guideline version 6 in the coronavirus disease diagnosis and treatment. Retrieved from http://www.nhc.gov.cn/xcs/zhengcwj/202002/8334a8326dd94d329df351d7da8aefc2.shtml. Accessed March 11, 2020. 6. Health Commission of the People’s Republic of China.(2009). The guideline for pathogens isolated operations in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/200904/40116.shtml. Accessed March 11, 2020. 7. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 8. Health Commission of the People’s Republic of China.(2017). The guideline for prevention and control of hospital acquired infections of airborne pathogens. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201701/7e0e8fc6725843aabba8f841f2f585d2.shtml. Accessed March 11, 2020. 9. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 10. Health Commission of the People’s Republic of China.(2012). The standardization for sterilization techniques in hospital. Retrieved from http://www.nhc.gov.cn/wjw/s9496/201204/54510.shtml. Accessed March 11, 2020. 11. Katona P. Bioterrorism Preparedness: Generic Blueprint for Health Departments, Hospitals, and Physicians. Infectious Diseases in Clinical Practice. 2002;11(3):115-122. Accessed March 11, 2020. 16 Figure Legends Figure 1. Diagram of the layout of our radiology department was divided into four areas: contaminated (shaded in black), semicontaminated (shaded in dark gray), buffer (shaded in light gray), and clean areas (shaded in white). The contaminated area was separated from other areas by barriers. Figure 2. Diagram shows CT protocol for suspected and confirmed patients with COVID-19. Figure 3. Diagram shows CT protocol for regular patients. Abbreviations: COVID-19: coronavirus disease 2019 CT: computed tomography DR: digital radiography EMICT: emergency management and infection control team NHC: National Health Commission PACS: picture archiving and communication system SARS: severe acute respiratory syndrome Sentence Summary With severe infectious diseases such as COVID-19, the highest level of infection control measures must be implemented, collaboration with the radiology departments of other hospitals be needed, and social media be employed. Take-home points 1. To response to a community infection emergency, a special emergency management team needs to be setup at the departmental level to implement infection containment and control procedures that continues to allow the imaging examination and imaging diagnosis of those with suspected infection, and to prevent intra-departmental spreading of infection (EMICT). 2. Infection control measures, such as reconfiguration of department areas, personal protection and anti-infection training of all staff, standardized procedures including contact minimization for chest CT and DR examinations, and timely disinfection of CT and DR examination rooms, should be implemented properly. 3. If there are more than one scanner in a hospital, only one of them should be assigned to suspected cases.	How many people have come in contact and how many of these are in observation?	2,465	677,243 people have been identified as having had close contact with infected patients of whom13,701	4,931
2,522	Identification of COVID-19 Can be Quicker through Artificial Intelligence framework using a Mobile Phone-Based Survey in the Populations when Cities/Towns Are Under Quarantine https://doi.org/10.1017/ice.2020.61 SHA: 83c96f2a481be06a5c58552cbad2ca67ce789dc2 Authors: Vazquez, Arni S.R. Srinivasa Rao; Jose A. Date: 2020 DOI: 10.1017/ice.2020.61 License: cc-by Abstract: We are proposing to use machine learning algorithms to be able to improve possible case identifications of COVID-19 more quicker when we use a mobile phone-based web survey. This will also reduce the spread in the susceptible populations. Text: Emerging and novel pathogens are a significant problem for global public health and technology can assist in faster identification of possible cases to bring timely interventions. This is especially true for viral diseases that are easily and readily transmissible and have asymptomatic infectivity periods. The novel Coronavirus (SARSCoV2) described in December 2019 has resulted in major quarantines throughout the world, including major cites, villages and public areas throughout China [1] [2] [3] to prevent further spread. As of February 25 th 2020, the World Health Organization's situational data indicates that there were about 77780 confirmed cases, including 2666 deaths due to COVID-19, including cases in 25 countries [4] . The majority of the deaths reported so far have been in China only [5] . Organization have issued interim guidelines in order to protect the population, and to attempt to prevent the further spread of COVID-19 from infected individuals [6] . Since cities and villages throughout China are unable to accommodate such large numbers of infected individuals, and be able to maintain the quarantined. China has built several new hospitals in an attempt to manage the infected individuals [7] . It is imperative that we evaluate novel models in an attempt to control the rapidly spreading virus [8] . In order to reduce the time to identification of a person under investigation (PUI) for the COVID-19 infection, and the rapid isolation of this individual, we propose to collect the basic travel history along with the more common manifestations using a phone-based online survey. Such collected data can be used to assist in the preliminary screening and early identification of possible COVID-19 infected individuals. Thousands of data points are able to be collected and processed through an artificial intelligence (AI) framework which can ultimately evaluate individuals that may be infected and stratify them into no-risk, minimal-risk, moderate-risk, and high-risk of being infected with the virus. The identification of the high-risk cases can then be quarantined earlier, thus decreasing the chance of spread. Table 1 is inserted here. See Appendix I for the details on the steps involved in data collection on all the respondents independent of whether or not they think they are infected. The AI algorithm described in Appendix II is to identify possible case identifications and send alerts to the nearest health clinic as well as to the respondent for an immediate health visit, we call this as an "alert for health check recommendation for COVID-2019. In case the respondent is unable to commute to the health center, the health department can then send an alert to a mobile health unit so they can then do doorto-door assessments and even testing for the virus. This generates alert for mobile health check recommendation for 2019-nCoV (MHCRC). If a respondent does not have an immediate risk of having symptoms or signs related to the viral infection, then the AI-based health alert will be sent to the respondent to notify them that there is no current risk of COVID-2019. Figure 1 summarizes the outcomes of data collection and identification of possible cases. The data recorded in step 5 of the algorithm using signs and symptoms will be collected prior to both the groups who have received alerts HCRC or MHCRC (for possible identification and assessment) and NCRC (for non-identified respondents). These are explained in steps (iii) and (iv) in the Appendix II. The extended analysis proposed will help to understand if there is any association with different sociodemographic variables and the manifestations such as fever and signs and lower respiratory infections, including cough and SOB in individuals defined as either with and without possible infection. Applications of AI and deep learning argued to be useful tools in assisting diagnosis and treatment decision making [10] [11] . There were studies which promoted disease detection through AI models [12] [13] [14] [15] . Use of mobile phones [16] [17] [18] [19] and web based portals [20] [21] have been tested successfully in health related data collection. However, one need to apply such techniques in a timely way for faster results. Apart from cost-effectiveness, the proposed modeling will be of great assistance in identifying and controlling when populations are closed due to virus spread. In addition to these, our proposed algorithm can be easily extended to identify individuals who might have any mild symptoms and signs. We have developed our data collection criteria based on CDC's Flowchart to Identify and Assess 2019 Novel Coronavirus [9] and added additional variables for the extended utility of our efforts in identifying infected and controlling the spread (see Table 1 ). Let be the outputs recorded during the data collection steps 1 (ii) If the set of identifiers, , for is equal to one of the elements of the set then send HCRC or MHCRC to that respondent, else proceed to the test criteria (iv). If is equal to one of the elements of the set , for then the respondent will be sent an NCRC alert. (iv) If is equal to one of the elements of the set , then the respondent will be sent an NCRC alert. Comparison of test criteria results of (iii) and (iv) with their corresponding geographic and socio-demographic details will yield further investigations of signs and symptoms Suppose we define two events and using the sets and as below: : out of responded cases are identified through the algorithm out of have responded to the survey. The conditional probability of the event given the event , say, is computed as	When was the novel Coronavirus first reported?	1,219	December 2019	974
1,714	Overview of the 3rd isirv-Antiviral Group Conference – advances in clinical management https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280814/ SHA: f7bb1f005066cb4930f83cde4cdc1ff3fe411def Authors: Hurt, Aeron C; Hui, David S; Hay, Alan; Hayden, Frederick G Date: 2014-11-15 DOI: 10.1111/irv.12293 License: cc-by Abstract: This review highlights the main points which emerged from the presentations and discussions at the 3rd isirv-Antiviral Group Conference - advances in clinical management. The conference covered emerging and potentially pandemic influenza viruses and discussed novel/pre-licensure therapeutics and currently approved antivirals and vaccines for the control of influenza. Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS-CoV, respiratory syncytial virus (RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted. Text: Recurrent infections by influenza and other respiratory viruses contribute enormously to the burden of human disease and (emergent) sporadic zoonotic infections, such as by influenza H7N9 and H5N1 and Middle East respiratory syndrome (MERS) coronavirus, pose a constant threat of a new global epidemic. Despite extensive knowledge of the viruses and their interaction with the host, there is little in our armoury of vaccines and therapeutics to combat this perpetual onslaught. The 3rd isirv Antiviral Group conference on Influenza and Other Respiratory Virus Infections: Advances in Clinical Management, convened in Tokyo, Japan on 4-6 June 2014, attracted 188 clinicians, public health specialists and medical scientists from 34 countries to present their recent research and discuss various aspects of the impact of respiratory viruses in different patient groups/settings and in different regions of the world. The programme 1 focused on the latest advances in the mitigation and clinical management of influenza and other respiratory virus disease, and the successful use of antivirals (and vaccines) against seasonal and pandemic influenza, particularly in Japan, as well as the development/assessment of novel antiviral agents. This overview highlights some of the main points which emerged from the presentations (both oral and poster) and associated discussion. In recent years, an increasing number of cases of novel animal influenza A viruses infecting humans have been reported. These include multiple avian influenza A virus subtypes, in particular H5N1 and H7N9, and swine-origin H3N2v. Reasons for this increase include both social factors, for example, increased human populations living in close proximity to animals and increased surveillance and diagnostic testing. Risk assessment tools have been developed by many authorities around the world (e.g. the European CDC, US CDC, USAID and WHO) to assist in predicting the likelihood that a particular virus will emerge and its associated impact, as well as prioritising the development of candidate human vaccine viruses. 2 These tools allow continual reassessment as new data become available and provide an objective, transparent process with which to make resource allocation and pandemic planning decisions. In February 2013, China detected the first human cases of H7N9 infection in severely ill patients with pneumonia. 3 As of May 22, 2014 , there have been 446 confirmed H7N9 cases in China resulting in 163 deaths. 4 The cases have occurred mainly during two waves (weeks [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 2013 and week 40, 2013week 20, 2014) , 4 of which 85% had prior exposure to poultry or contaminated live poultry markets. The median time from poultry exposure to disease onset was 5 days, whereas the median time from illness onset to hospital admission, ARDS development, antiviral therapy and death was 5, 6Á8, 7 and 14 days, respectively. 5 Closure of live poultry markets has markedly reduced the risk of H7N9 infection. Across nine areas in the two most affected provinces in China, modelling analysis estimated that the effectiveness of market closure was 97% (95% CI: 89%, 100%). 6 A retrospective serological study of blood specimens taken in January-May and October-November in 2012 from 1544 subjects who worked in live poultry markets, farms, slaughter houses or kept backyard poultry revealed no evidence of H7N9 infection, 7 indicating widespread population susceptibility and lack of prior circulation of antigenically related viruses. Multiple family clusters have been reported, but no sustained human-to-human transmission, with studies demonstrating a very low detection of virus or specific antibody in close contacts (0Á34% and 0Á2% in the first and second waves, respectively) and healthcare workers of positive cases. 8 In both the first and the second waves, the majority of the patients hospitalised with H7N9 infection were older men (median age, 62 and 58 years, respectively with an overall male/female ratio of 2Á2:1) and the case fatality was similar (32% and 39%, respectively). Pre-existing medical conditions occurred in >60% of these cases. The prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, while the most striking laboratory findings were marked lymphopenia and thrombocytopenia. Elevated cytokine levels have been observed in patients and such excessive cytokine responses may contribute to the clinical severity of H7N9 infection. 9 Originating from reassortment events involving at least three avian influenza viruses, H7N9 viruses with multiple genotypes continue to emerge on a more frequent basis in 2014. Many viruses isolated from humans contain the E627K amino acid substitution in the polymerase PB2 component, associated with mammalian adaptation, and the G186V and Q226L substitutions in the haemagglutinin (HA) that are associated with dual receptor binding to both a2,3 and a2,6-linked sialic acid receptors. 10 All H7N9 viruses from the outbreak to date are antigenically similar to the (original) candidate vaccine strain. Prototype inactivated whole particle H7N9 vaccines have been investigated in macaques and shown to induce good antibody responses that significantly reduced the number of days of virus shedding in experimentally infected animals. Treatment of H7N9-infected patients with neuraminidase inhibitors (NAIs), including intravenous (IV) peramivir or zanamivir, 11 appears to have been beneficial even when therapy was started late, although emergence of oseltamivir resistance has been associated with poor clinical outcomes. 12 All H7N9 viruses are amantadine-resistant due to the S31N substitution in the M2 ion channel protein, while viruses containing the R292K substitution in neuraminidase (NA), which confers resistance to both oseltamivir and peramivir (>1000-fold rise in IC 50 ) and reduced susceptibility to zanamivir and laninamivir (50-and 25-fold rises in IC 50 , respectively), have been reported in six cases. Two of these patients with severe H7N9 infection requiring extracorporeal membrane oxygenation (ECMO) also received systemic corticosteroid treatment leading to treatment failure and a poor clinical outcome. 12 The replication and transmission of H7N9 viruses containing the R292K NA mutation have been shown to be comparable to those of wild-type H7N9 viruses in guinea pigs, 13 and in ferrets following both contact and non-contact exposure. However, the wild-type virus did outgrow the R292K-resistant strain in some ferrets over the course of the infection. 14 Interestingly, the R292K variant appeared to be the dominant virus in ferret lung lobes, while in nasal turbinates, the wild-type virus was predominant. Therefore, it appears that the R292K mutation causes less fitness loss in H7N9 virus than in seasonal H3N2 viruses. 13 A new reassortant genotype of H5N1 containing the HA and NA genes from clade 1.1.2 and the internal genes from clade 2.3.2.1 emerged during 2013 and was associated with the highest number of cases (n = 26) and deaths (n = 14) in Cambodia. 15 Globally since 2003, there have been 650 confirmed H5N1 cases and 386 deaths reported in humans, 16 with most infections in the last 2 years being in children. Human-to-human transmission remains extremely rare based on virological and serological data from analysis of close contacts of confirmed cases in Cambodia, Thailand and Vietnam. 17 A study of household transmission patterns in Indonesia has shown that the overall household attack rate was 18Á3% and the secondary attack rate was 5Á5%, independent of household size. 18 Oseltamivir therapy appears to reduce mortality when administered within 8 days of H5N1 illness onset, although earlier treatment is more effective, highlighting the need for early patient diagnosis. 19 Early initiation of oseltamivir was particularly effective in reducing mortality in H5N1 patients without respiratory failure (odds ratio, 0Á17; P = 0Á04), whereas those requiring ventilatory support at the time of oseltamivir initiation were more likely to die. 20 A study of the risk factors for mortality related to H5N1 identified age, country, per capita government health expenditure and delay from symptom onset to hospitalisation as the key parameters, highlighting the importance of early diagnosis, treatment and supportive care. 21 High-dose systemic corticosteroids (SC) are associated with worse outcomes in H5N1 patients. 22 One human case of avian H5N6 was recently detected in China; the virus was a reassortant that contained seven genes from H5N1 and the NA gene from an H6N6 virus circulating in ducks. 23 China has also reported the detection of three human infections, two fatal, with avian H10N8 viruses that contain the internal genes from H9N2, as does H7N9. 24 Like the H7N9 virus, the H10N8 virus has low pathogenicity in poultry and is therefore difficult to detect in birds. Burden in target populations Pregnant women and infants have an increased risk of complications following influenza infection. Globally, significant numbers of pregnant women died during the 2009-2010 pandemic, but no maternal mortality occurred in Japan. 25 Through education campaigns directed at pregnant women and healthcare professionals, 67% of pregnant women were vaccinated against H1N1pdm09 resulting in an infection rate among pregnant women in Japan of 3Á5% compared to the overall infection rate in the population of 12%. 26 Of those pregnant women who were infected with H1N1pdm09 in Japan, 95% were treated with antivirals, and importantly, 88% of those were treated within 2 days of symptom onset. 25 In Mongolia, a prospective cohort study during 2013-2014 found that influenza-like illness (ILI) was detected in 17Á9% of pregnant women, of whom the majority tested positive for influenza A, with substantially lower influenza B and respiratory syncytial virus (RSV) infection. 27 During the same period, ILI was detected in 30Á9% of infants <6 months of age, with an even spread of influenza A, influenza B and RSV. 27 The influenza burden in children in a rural Indian community was found to be substantial with 11Á6% of ILI cases being caused by influenza A or B viruses. 28 These findings underscore the importance of maternal immunisation. 29 Transmission patterns Sequence analysis of influenza viruses isolated from students on a Singapore University Campus provided insights into the chain of transmission, showing that 62% of 32 viruses were highly similar, demonstrating that the majority of transmission was occurring on the university campus rather than from infections outside. 30 The effectiveness of surgical masks, hand hygiene and health education investigated in households in Hong Kong and Bangkok detected no significant difference in attack rate in cohorts using one of these interventions. 31 Further analysis of the data enabled some insights into the relative importance of aerosol, large droplet and contact transmission within the households. For influenza A infections, aerosol transmission appeared to be the most common route, whereas contact transmission caused the highest number of influenza B infections. 31 Use and effectiveness Neuraminidase inhibitors are commonly used for the treatment of influenza in Japan, typically following a positive result from a point-of-care (POC) test. During the 2009-2010 pandemic, over 20 million POC test kits were shipped to hospitals and clinics in Japan to enable rapid diagnosis, and 89% of treated cases were administered NAIs within 48 hours of symptom onset. 32 In Japan during 2013, oseltamivir and laninamivir each represented 40% of NAIs used, while zanamivir (15%) and peramivir (5%) use was considerably less. NAI effectiveness has been assessed in numerous observational studies in Japan. Oseltamivir effectiveness is significantly reduced in patients with delayed treatment, and duration of fever and viral shedding is longer in treated patients with influenza B compared to influenza A virus infections. 33, 34 The reduced effectiveness against influenza B viruses was also observed in zanamivir 33, 35 and laninamivir 35 trials. To determine whether NAIs reduced mortality during the 2009-2010 pandemic, data were compiled on 29 234 patients hospitalised with confirmed A(H1N1)pdm09 infection. 36 Compared with no treatment, NAI treatment was associated with significantly reduced mortality, with early treatment also showing a reduced risk of mortality compared to late treatment. Although there was no significant clinical effect when comparing late treatment with no treatment in hospitalised patients, there was a significant benefit in treating patients who arrive late into intensive care units. 36 In a household prophylaxis study, inhaled laninamivir given for either 2 or 3 days reduced the illness rate within households to 3Á9% and 3Á7%, respectively, compared to 16Á9% in households given a placebo. 37 A ferret model of oseltamivir prophylaxis has shown that while morbidity was significantly reduced, the prophylaxis regimes did not prevent infection nor significantly reduce virus load. 38 Resistance Although all four NAIs are sialic acid analogues, they have subtle differences in chemical structure and binding properties. Consequently, resistance patterns vary across NAIs. The most commonly detected NA substitution causing NAI resistance in N1-containing influenza viruses is H275Y, which confers resistance to oseltamivir and peramivir, but not to zanamivir and laninamivir. This resistance mutation became fixed in seasonal H1N1 viruses circulating in 2008-2009. 39 A late 2013 cluster of H1N1pdm09 viruses containing the H275Y substitution was detected in 38 (39%) of 97 H1N1pdm09 viruses from community patients not receiving NAIs in Sapporo, Japan, 40 reminiscent of a similar cluster of oseltamivir-resistant H1N1pdm09 viruses in community patients in Australia in 2011. 41, 42 Importantly, both sets of viruses contained permissive NA mutations (V241I and N369K) that have been shown in ferret studies to offset the destabilising and negative effect of the H275Y NA mutation. 43 In hospitalised influenza patients being treated with intravenous zanamivir, next-generation sequencing has been utilised to identify minor resistant virus populations. A total of five NA substitutions were identified in different viruses, including E119K and E119D; however, all apart from E119D were present in such low proportions that they could not be detected by Sanger 'population' sequencing methods. 44 The effects of various mutations in catalytic and framework residues of influenza B NA were investigated using reverse genetics and a range of functional assays. Four substitutions (D198E, I222T, H274Y and N294S) conferred reduced susceptibility to oseltamivir, while three substitutions (E119A, D198Y and R371K) caused highly reduced inhibition by oseltamivir, zanamivir and peramivir. 45 Two of these variants (H274Y, E119A) had in vitro replication fitness comparable to the NAI-susceptible viruses. To date, these substitutions have only been detected on rare occasions in circulating influenza viruses. An intravenous formulation of zanamivir showed both virological and clinical effectiveness without safety concerns in patients hospitalised with influenza in Japan. 46 A range of new adamantane derivatives have good antiviral activity in vitro and in animal models against H1N1pdm09 and H3N2 viruses that contain the S31N M2 ion channel substitution that confers resistance to amantadine. 47 Favipiravir is a novel pyrazinamide molecule that inhibits replication of various RNA viruses, including influenza types A, B and C (including oseltamivir-resistant strains), and has recently been licensed in Japan for the control of novel or reemerging influenza viruses. Its triphosphate metabolite is an RNA polymerase inhibitor which disrupts virus genome replication; synergy with oseltamivir has been demonstrated in pre-clinical models. 48 A phase II study in the US has shown that a twice-daily regimen decreased the titre and time to cessation of virus shedding, and had a significant benefit in reducing clinical symptoms (NCT01068912; www.clinicaltrials.gov). Subsequent phase III studies are currently ongoing (NCT02008344 and NCT02026349). A neutralising monoclonal antibody (MHAA4549A) which binds to the HA stalk of influenza A viruses in both group 1 and group 2 HA subtypes has been effective when given up to 72 hours post-infection in mice and ferrets infected with H5N1. 49 Phase I and IIa trials in humans showed that the antibody was well tolerated, had a mean half-life of 21Á9 days and was effective as therapy at high doses in experimentally infected volunteers (NCT01877785). Upcoming placebo-controlled phase IIb trials will target hospitalised influenza patients requiring oxygen and compare the combination of the monoclonal antibody with oseltamivir to oseltamivir monotherapy (NCT01980966). Other broadly neutralising antibodies against multiple clades of H5N1 have been generated by glycan masking of key HA antigenic residues to direct antibody responses to the more conserved stem region of the HA. 50 FluPep, a novel peptide that prevents virus entry into cells, has been shown in mouse studies to be effective in reducing virus titres in lungs, inflammatory cytokines and mortality. 51 Fludase (DAS-181) is a host-targeted therapeutic agent that removes sialic acid from cellular receptors in the respiratory tract, thus preventing influenza virus binding. Delivered topically, it is effective in animal models of lethal H5N1 and H7N9 infection, including a NAI-resistant R292K H7N9 variant. 52 In a phase 2 RCT, inhaled DAS181 reduced pharyngeal viral replication in uncomplicated influenza but did not reduce nasal virus loads or improve clinical outcomes. 53 Another receptor-targeted approach is the development of multivalent sialic acid-binding proteins; 54 a single administration 7 days pre-infection resulted in the protection of 80-100% of mice from lethal H7N9 challenge. 55 Apart from blocking sialic acid, the compound appears to stimulate the expression of pro-inflammatory mediators, thereby 'preparing' the immune system for subsequent influenza infection. When delivered 24 hours post-infection, protection was, however, only 20-40%. 55 Although drug resistance is considered less likely to occur with host-directed therapies, escape mutants have developed rapidly following exposure to a host-directed vacuolar ATPase-inhibiting drug. 56 Furthermore, following serial passage of different viruses in the presence of bafilomycin A1, two HA mutations were selected (A19T and S210N) which resulted in reduced drug susceptibility and increased virulence in mice. 56 Multiple influenza vaccine effectiveness (IVE) studies have used the control test negative design approach to estimate IVE during early and late phases of influenza seasons, the 2009 pandemic, and by age or target groups. Typically, IVE estimates range from 40% to 60% each season. 57 Future studies will investigate IVE with respect to the type of influenza vaccine used, whether IVE differs between the start and end of the season and the effect of previous vaccination. In Japan in 2013/14, IVE for influenza A in children aged 1-5 years averaged 72% (95% CI 64-79), dropped to 48% (95% CI 31-61) in children aged 6-12 years and was not apparent against influenza B in any age group (À1%, 95% CI À19 to 14). 58 Influenza vaccine effectiveness is known to be lower in adults over 65 years of age, a group that accounts for >60% of seasonal influenza-related hospitalisations and >90% of influenza-related deaths. In an effort to improve IVE in the elderly, recent RCTs have investigated the use of adjuvants, intradermal injection and higher doses of antigen. While the use of AS03-adjuvanted influenza vaccine was only moderately superior to non-adjuvanted vaccine in the elderly, 59 the use of a high-dose vaccine containing four times the standard level of HA (60 lg per virus) did result in improved effectiveness compared to the standard dose vaccine. 60 For vaccine manufacturers, generating high-growth reassortants of certain circulating viruses can be challenging. A recent study used random mutagenesis of PR8 and selection of high-growth clones in MDCK and Vero cells to derive a high-growth version of PR8. 61 Reassortment of the highgrowth PR8 virus with the HA/NA of either H5N1, H7N9 or seasonal influenza viruses showed that yields significantly exceeded equivalent reassortants that contained the internal genes of the 'normal' PR8 virus. 61 As of July 2014, the number of confirmed cases of MERS-CoV has exceeded 830, with at least 288 associated deaths. 62 The majority of cases have involved patients with comorbidities (76%) and are predominately males (63%) with a median age of 47. 63, 64 Fewer than 25% of patients have reported contact with animals including dromedary camels, which have been shown to be one likely animal reservoir based on sero-positivity and detection of MERS-CoV. 65 More than 25% of the infections have been in healthcare workers, and the large number of nosocomial infections is likely due to inadequate infection control in hospitals plus enhanced surveillance that has detected a substantial number of mild or asymptomatic infections. 63 Outside hospital, the burden of disease is likely to be larger than has been reported. 66 Serological analysis of several UK patients found a rapid rise in antibodies from day 10, and that titres were maintained for at least 300 days post-infection. Anti-S (spike glycoprotein) antibodies are responsible for virus neutralisation. Importantly for serological analyses, patients who experience only mild disease may mount only a modest serological response. 67 Sequential samples from three cases involved in a chain of transmission were extensively analysed using next-generation sequencing. 68 Various minority variants were detected, of which some were transient while others were transmitted, and there was evidence of variation in frequency of some variants in different body compartments. Various therapeutic options have been investigated for the treatment of MERS-CoV, but no therapy of proven value currently exists. The use of SC was associated with adverse outcome in SARS 69 and is not recommended for MERS-CoV. Many agents have shown inhibitory effects against MERS-CoV in cell culture including interferon +/À ribavirin, cyclosporine A, mycophenolic acid, chloroquine and lopinavir. 70 Interferons, lopinavir, mycophenolate, possibly alisporivir and combinations are reasonable choices for testing in controlled clinical trials. Exploratory post hoc metaanalysis of studies related to SARS and severe influenza has shown a significant reduction in mortality following convalescent plasma treatment compared to placebo or no therapy (odds ratio 0Á25; 95% CI 0Á14-0Á45). 71 Thus, the early use of virus-specific neutralising antibodies in the form of convalescent plasma and monoclonal or polyclonal neutralising antibodies for treatment of MERS-CoV has the highest likelihood of clinical benefit. 64 Modalities with risks likely to exceed benefits include SC, ribavirin monotherapy and IVIG. 72 Respiratory syncytial virus (RSV) Respiratory syncytial virus disproportionately impacts children in low-income countries. 73 Almost all children will have been infected with RSV by their 2nd birthday, and it is the number one cause of hospitalisation of infants in the US, causing 10 times more infant deaths than influenza. 74 In addition, RSV infects 3-10% of adults annually and accounts for 5-15% of community acquired pneumonia (CAP) and 9-10% of hospitalisations, 75 a burden of disease that approaches that caused by influenza. In a study, conducted in Hong Kong, of 607 hospitalised adults with RSV, 40% had pneumonia and 70% required supplementary oxygen; mortality rates and duration of hospital stay were similar to those observed for influenza patients. 75 Approximately, 15% of hospitalised RSV patients had bacterial superinfections. Although corticosteroids were used to treat 38% of patients, treatment had no benefit on clinical outcome, and instead increased bacterial secondary infections and caused a longer duration of illness. 75 RSV replication appears prolonged in patients with comorbidities and LRT complications. Palivizumab prophylaxis of premature infants of <6 months of age has been shown to reduce hospitalisation due to RSV by 55%. 76 Preventing RSV during infancy has been associated with reduction of wheezing later in life. 77 Trials of other monoclonal antibodies have typically shown that they do not achieve superiority compared to palivizumab and therefore do achieve licensure. Furthermore, treatment with neutralising monoclonal antibodies does not appear to reduce virus load or disease severity in hospitalised infants. 78 Alternative options for RSV therapy to be assessed in future clinical trials include inhaled nanobodies, aerosolised peptides, nucleoside analogues and RNA-interference molecules. Human rhinoviruses (HRV) usually cause mild acute respiratory infections, but on occasions can also cause more severe respiratory infections, including exacerbations of asthma and COPD. Of 115 Japanese children with asthma, a respiratory virus was detected in 86%, of which HRV (n = 36) or RSV (n = 47) were most common. 79 Ex vivo bronchial epithelial cells from people with asthma are more susceptible to HRV infection, due to deficient induction of IFN-b and IFN-lambda. In a study of 147 asthmatics on inhaled corticosteroid therapy, with a history of virusassociated exacerbations, patients were randomised to 14-day treatment with inhaled IFN-b or placebo within 24 hours of developing cold symptoms. Patients who received IFN-b had enhanced morning peak expiratory flow recovery, reduced need for additional treatment and boosted innate immunity as assessed by blood and sputum biomarkers. In an exploratory analysis of a subset of more difficult-to-treat asthma (n = 27 IFN-b; n = 31 placebo), worsening of symptoms increased significantly in the placebo group, but was prevented by IFN-b (P = 0Á004). 80 A picornavirus-specific antiviral, vapendavir, was found to reduce symptom scores, lower bronchodilator puffer use and reduce viral load in asthma patients with an URTI due to HRV. 81 Diagnostics Point-of-care tests that can deliver a result in 15 minutes have been available in many countries for the last decade, but while having good specificity, the sensitivity has typically been poor, ranging from 10% to 80% compared to PCR or culture. Their use in emergency departments of hospitals can result in reduced unnecessary antibiotic use and an increased likelihood of discharge. Newer immunofluorescence-based POC tests with improved sensitivity are being developed. In addition, the Quidel Sofia POC test may be linked via the internet such that results can be reported in real-time to central databases. Other POC tests are using photographic silver amplification immunochromatography technology to increase sensitivity. 82 PCR remains the gold standard for virus diagnostics with an ability to be rapid, sensitive, specific and to identify a wide range of pathogens via different assays. The ability to multiplex multiple pathogen targets allows costs to be reduced in a diagnostic setting. New closed-system technologies which involve only minimal hands-on time (a few minutes) and that conduct both automated nucleic acid extraction and PCR for multiple pathogens are now available, but are currently limited for clinical diagnostic purposes due to low-throughput capabilities. 83 Next-generation sequencing technologies and PCR-based analyses with increasing sensitivity both offer considerable scope in diagnosis, although our current understanding of the clinical impact of pathogens at low levels or the presence of variants as minor virus populations is limited. Providing low-cost, sensitive assays for diagnosing respiratory virus infections in low/middle-income countries is challenging, but has the potential to improve treatment and avoid unnecessary antibiotic use in these regions. Repurposed drugs for respiratory viral infections Nitazoxanide (NTX) is an antiparasitic agent approved for Giardia and Cryptosporidium infections that also inhibits replication in vitro of influenza and other respiratory viruses. 84 Treatment with NTX 600 mg twice daily for 5 days was associated with a reduction in the duration of symptoms in participants with acute uncomplicated influenza. 85 In a subset analysis of 238 patients with no confirmed virus infection, treatment with NTX 600 mg also led to a shorter time to alleviation of symptoms in comparison to placebo (88Á4 versus 105Á7 hours, P = 0Á02). 86 Systemic corticosteroids for respiratory virus infections A review of prospective observational studies has shown that SC increased the risks of mortality and morbidity (e.g. secondary infections, hospital-acquired pneumonia) in severe infection due to influenza A(H1N1)pdm09 especially with delayed antiviral therapy. 87 During SARS infections, a higher risk of avascular necrosis and prolonged virus shedding were observed in patients who had received highdose SC therapy. 88 It is therefore important to avoid the use of high-dose SC in severe respiratory viral infections outside the context of clinical trials. Larger trials are needed to resolve the uncertainty regarding the effect of early SC therapy in ARDS. Low-dose SC is indicated for management of refractory septic shock, 89 and a short course of SC is indicated for acute exacerbations of obstructive airway diseases (asthma, COPD) 90 Current evidence does not support a clinically relevant effect of systemic or inhaled glucocorticoids on admission or length of hospitalisation for acute viral bronchiolitis in infants and young children. 91 Respiratory syncytial virus, influenza viruses, parainfluenza (PIV) viruses and adenoviruses (AdVs) cause the most serious disease in immunocompromised hosts, but other respiratory viruses are becoming increasingly appreciated as a cause of both upper and lower respiratory tract disease. The potential for these viruses to cause lower respiratory tract infections (LRTI) after transplantation varies. Human metapneumovirus infections have similar outcomes to RSV infection in hematopoietic stem cell transplant (HSCT) recipients, including potentially severe and fatal pneumonia. HRV and coronavirus infections are very frequent in transplant recipients, but severe lower respiratory tract disease is uncommon. In a prospective study of 112 lung transplant recipients, the virus infection rates upon screening, routine and emergency visits were 14%, 15% and 34%, respectively. Picornaviruses were identified most frequently in nasopharyngeal (85/140; 61%) and BAL specimens (20/34; 59%). Asymptomatic virus carriage, mainly of picornaviruses, was found at 10% of screening visits. Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels. The hospitalisation rate was 50% for influenza and PIV and 16Á9% for other viruses. Acute rejection was not associated with virus infection. 92 The risk factors for severe LRTI among transplant recipients include early onset post-transplant (<3 m), steroid boluses, young children (<1 year), chronic GVHD, lymphopenia/lymphodepletion and allogeneic HSCT patients. 92 Influenza Immunocompromised patients with influenza exhibit more complications, longer virus shedding and more antiviral resistance, while often demonstrating milder clinical symptoms and signs on initial clinical assessment. 93 Influenza A (H1N1)pdm09 viruses have the potential for rapid emergence of oseltamivir resistance and causing severe morbidity, particularly in immunocompromised patients with lymphopenia and delayed antiviral therapy. 94 Influenza viraemia may serve as a marker for overall poor outcome with increased risk of progression to LRTI, hypoxaemia, respiratory failure and death. Influenza RNA in blood (viraemia) was detected in nine of 79 (11Á4%) HSCT recipients with influenza. Among patients with LRTI, viraemia was associated with increased hazards of overall as well as influenzaassociated death (hazard ratio 3Á5, 1Á1-12). 95 In 143 HSCT recipients with documented seasonal influenza infection, treatment with high-dose corticosteroids was associated with a trend towards prolonged virus shedding [(OR), 3Á3; 95% CI 1Á0-11; P = 0Á05], whereas antiviral therapy initiated to treat upper respiratory tract infection (URTI) was associated with fewer cases of LRTI (OR, 0Á04; 95% CI, 0-0Á2; P < 0Á01) and fewer hypoxaemia episodes (OR, 0Á3; 95% CI, 0Á1-0Á9; P = 0Á03). 96 In view of the risks of prolonged replication and drug resistance emergence, 97 a longer duration and a higher NAI dose may be beneficial. Early therapy was consistently demonstrated to have improved outcomes. [98] [99] [100] [101] Other treatment options under study are the use of triple combination therapy with amantadine, oseltamivir and ribavirin, 102 or intravenous peramivir 103 or zanamivir. 104 Therapy of influenza in lung transplant recipients is associated with a reduced risk of developing bronchiolitis obliterans syndrome. 105 Parainfluenza DAS181 is inhibitory for PIV and influenza viruses, including those resistant to the amantadine and NAIs 106 and may be effective in treating immunocompromised patients with severe PIV lung disease. 107 In a study of four severely immunocompromised children with PIV disease treatment with DAS181 for 5-10 days, by dry powder inhalation or nebulisation, was well tolerated. Transient increase in serum alkaline phosphatase, liver function and coagulation tests were observed, but nasal wash virus loads were reduced in all patients within 1 week with improved clinical features. 108 In a RCT of lung transplant recipients with RSV infection, the incidence of new or progressive bronchiolitis obliterans syndrome at day 90 was significantly reduced in 16 patients who received a small interfering RNA against the RSV Ngene (ALN-RSV01) compared with placebo (n = 8) (6Á3% versus 50%, P = 0Á027). 109 In a larger follow-up multicentre phase IIb study, treatment with ALN-RSV01 showed a greater than eightfold reduced risk in developing bronchiolitis obliterans syndrome at day 180. 110 Adenovirus is a serious, often fatal infection in immunocompromised patients, especially in HSCT recipients. The control of AdV is mostly T-cell mediated, and therefore, patients who have received T-cell suppressive regimens are at an increased risk for AdV infection. The annual incidence of AdV infections in HSCT recipients ranges from 5% to 50%, and is increasing, likely due to increased use of T-celldepleted allografts and cord blood as source. The mortality rate is up to 80%. 111 Brincidofovir (BCV; formerly CMX-001) is an orally bioavailable lipid-conjugate of cidofovir (CDV) that provides high intracellular concentrations of CDV diphosphate with a long intracellular half-life (up to 4-6Á5 days). BCV is 65-fold more potent against AdV than CDV in vitro with a low risk of myeloidor nephrotoxicity, but gastrointestinal side effects are more common. 112 In a retrospective study of 13 immunocompromised patients given BCV for AdV disease after failing or intolerance to i.v. cidofovire nine patients (69Á2%) demonstrated a virological response (VR), which was defined as a 99% drop from baseline or undetectable AdV DNA in serum by week 8. Patients with VR had longer survival than those without VR (median 196 days versus 54Á5 days; P = 0Á04). 113	When did China detect the first human case of H7N9 infection?	5,320	February 2013	3,243
1,714	Overview of the 3rd isirv-Antiviral Group Conference – advances in clinical management https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280814/ SHA: f7bb1f005066cb4930f83cde4cdc1ff3fe411def Authors: Hurt, Aeron C; Hui, David S; Hay, Alan; Hayden, Frederick G Date: 2014-11-15 DOI: 10.1111/irv.12293 License: cc-by Abstract: This review highlights the main points which emerged from the presentations and discussions at the 3rd isirv-Antiviral Group Conference - advances in clinical management. The conference covered emerging and potentially pandemic influenza viruses and discussed novel/pre-licensure therapeutics and currently approved antivirals and vaccines for the control of influenza. Current data on approved and novel treatments for non-influenza respiratory viruses such as MERS-CoV, respiratory syncytial virus (RSV) and rhinoviruses and the challenges of treating immunocompromised patients with respiratory infections was highlighted. Text: Recurrent infections by influenza and other respiratory viruses contribute enormously to the burden of human disease and (emergent) sporadic zoonotic infections, such as by influenza H7N9 and H5N1 and Middle East respiratory syndrome (MERS) coronavirus, pose a constant threat of a new global epidemic. Despite extensive knowledge of the viruses and their interaction with the host, there is little in our armoury of vaccines and therapeutics to combat this perpetual onslaught. The 3rd isirv Antiviral Group conference on Influenza and Other Respiratory Virus Infections: Advances in Clinical Management, convened in Tokyo, Japan on 4-6 June 2014, attracted 188 clinicians, public health specialists and medical scientists from 34 countries to present their recent research and discuss various aspects of the impact of respiratory viruses in different patient groups/settings and in different regions of the world. The programme 1 focused on the latest advances in the mitigation and clinical management of influenza and other respiratory virus disease, and the successful use of antivirals (and vaccines) against seasonal and pandemic influenza, particularly in Japan, as well as the development/assessment of novel antiviral agents. This overview highlights some of the main points which emerged from the presentations (both oral and poster) and associated discussion. In recent years, an increasing number of cases of novel animal influenza A viruses infecting humans have been reported. These include multiple avian influenza A virus subtypes, in particular H5N1 and H7N9, and swine-origin H3N2v. Reasons for this increase include both social factors, for example, increased human populations living in close proximity to animals and increased surveillance and diagnostic testing. Risk assessment tools have been developed by many authorities around the world (e.g. the European CDC, US CDC, USAID and WHO) to assist in predicting the likelihood that a particular virus will emerge and its associated impact, as well as prioritising the development of candidate human vaccine viruses. 2 These tools allow continual reassessment as new data become available and provide an objective, transparent process with which to make resource allocation and pandemic planning decisions. In February 2013, China detected the first human cases of H7N9 infection in severely ill patients with pneumonia. 3 As of May 22, 2014 , there have been 446 confirmed H7N9 cases in China resulting in 163 deaths. 4 The cases have occurred mainly during two waves (weeks [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 2013 and week 40, 2013week 20, 2014) , 4 of which 85% had prior exposure to poultry or contaminated live poultry markets. The median time from poultry exposure to disease onset was 5 days, whereas the median time from illness onset to hospital admission, ARDS development, antiviral therapy and death was 5, 6Á8, 7 and 14 days, respectively. 5 Closure of live poultry markets has markedly reduced the risk of H7N9 infection. Across nine areas in the two most affected provinces in China, modelling analysis estimated that the effectiveness of market closure was 97% (95% CI: 89%, 100%). 6 A retrospective serological study of blood specimens taken in January-May and October-November in 2012 from 1544 subjects who worked in live poultry markets, farms, slaughter houses or kept backyard poultry revealed no evidence of H7N9 infection, 7 indicating widespread population susceptibility and lack of prior circulation of antigenically related viruses. Multiple family clusters have been reported, but no sustained human-to-human transmission, with studies demonstrating a very low detection of virus or specific antibody in close contacts (0Á34% and 0Á2% in the first and second waves, respectively) and healthcare workers of positive cases. 8 In both the first and the second waves, the majority of the patients hospitalised with H7N9 infection were older men (median age, 62 and 58 years, respectively with an overall male/female ratio of 2Á2:1) and the case fatality was similar (32% and 39%, respectively). Pre-existing medical conditions occurred in >60% of these cases. The prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, while the most striking laboratory findings were marked lymphopenia and thrombocytopenia. Elevated cytokine levels have been observed in patients and such excessive cytokine responses may contribute to the clinical severity of H7N9 infection. 9 Originating from reassortment events involving at least three avian influenza viruses, H7N9 viruses with multiple genotypes continue to emerge on a more frequent basis in 2014. Many viruses isolated from humans contain the E627K amino acid substitution in the polymerase PB2 component, associated with mammalian adaptation, and the G186V and Q226L substitutions in the haemagglutinin (HA) that are associated with dual receptor binding to both a2,3 and a2,6-linked sialic acid receptors. 10 All H7N9 viruses from the outbreak to date are antigenically similar to the (original) candidate vaccine strain. Prototype inactivated whole particle H7N9 vaccines have been investigated in macaques and shown to induce good antibody responses that significantly reduced the number of days of virus shedding in experimentally infected animals. Treatment of H7N9-infected patients with neuraminidase inhibitors (NAIs), including intravenous (IV) peramivir or zanamivir, 11 appears to have been beneficial even when therapy was started late, although emergence of oseltamivir resistance has been associated with poor clinical outcomes. 12 All H7N9 viruses are amantadine-resistant due to the S31N substitution in the M2 ion channel protein, while viruses containing the R292K substitution in neuraminidase (NA), which confers resistance to both oseltamivir and peramivir (>1000-fold rise in IC 50 ) and reduced susceptibility to zanamivir and laninamivir (50-and 25-fold rises in IC 50 , respectively), have been reported in six cases. Two of these patients with severe H7N9 infection requiring extracorporeal membrane oxygenation (ECMO) also received systemic corticosteroid treatment leading to treatment failure and a poor clinical outcome. 12 The replication and transmission of H7N9 viruses containing the R292K NA mutation have been shown to be comparable to those of wild-type H7N9 viruses in guinea pigs, 13 and in ferrets following both contact and non-contact exposure. However, the wild-type virus did outgrow the R292K-resistant strain in some ferrets over the course of the infection. 14 Interestingly, the R292K variant appeared to be the dominant virus in ferret lung lobes, while in nasal turbinates, the wild-type virus was predominant. Therefore, it appears that the R292K mutation causes less fitness loss in H7N9 virus than in seasonal H3N2 viruses. 13 A new reassortant genotype of H5N1 containing the HA and NA genes from clade 1.1.2 and the internal genes from clade 2.3.2.1 emerged during 2013 and was associated with the highest number of cases (n = 26) and deaths (n = 14) in Cambodia. 15 Globally since 2003, there have been 650 confirmed H5N1 cases and 386 deaths reported in humans, 16 with most infections in the last 2 years being in children. Human-to-human transmission remains extremely rare based on virological and serological data from analysis of close contacts of confirmed cases in Cambodia, Thailand and Vietnam. 17 A study of household transmission patterns in Indonesia has shown that the overall household attack rate was 18Á3% and the secondary attack rate was 5Á5%, independent of household size. 18 Oseltamivir therapy appears to reduce mortality when administered within 8 days of H5N1 illness onset, although earlier treatment is more effective, highlighting the need for early patient diagnosis. 19 Early initiation of oseltamivir was particularly effective in reducing mortality in H5N1 patients without respiratory failure (odds ratio, 0Á17; P = 0Á04), whereas those requiring ventilatory support at the time of oseltamivir initiation were more likely to die. 20 A study of the risk factors for mortality related to H5N1 identified age, country, per capita government health expenditure and delay from symptom onset to hospitalisation as the key parameters, highlighting the importance of early diagnosis, treatment and supportive care. 21 High-dose systemic corticosteroids (SC) are associated with worse outcomes in H5N1 patients. 22 One human case of avian H5N6 was recently detected in China; the virus was a reassortant that contained seven genes from H5N1 and the NA gene from an H6N6 virus circulating in ducks. 23 China has also reported the detection of three human infections, two fatal, with avian H10N8 viruses that contain the internal genes from H9N2, as does H7N9. 24 Like the H7N9 virus, the H10N8 virus has low pathogenicity in poultry and is therefore difficult to detect in birds. Burden in target populations Pregnant women and infants have an increased risk of complications following influenza infection. Globally, significant numbers of pregnant women died during the 2009-2010 pandemic, but no maternal mortality occurred in Japan. 25 Through education campaigns directed at pregnant women and healthcare professionals, 67% of pregnant women were vaccinated against H1N1pdm09 resulting in an infection rate among pregnant women in Japan of 3Á5% compared to the overall infection rate in the population of 12%. 26 Of those pregnant women who were infected with H1N1pdm09 in Japan, 95% were treated with antivirals, and importantly, 88% of those were treated within 2 days of symptom onset. 25 In Mongolia, a prospective cohort study during 2013-2014 found that influenza-like illness (ILI) was detected in 17Á9% of pregnant women, of whom the majority tested positive for influenza A, with substantially lower influenza B and respiratory syncytial virus (RSV) infection. 27 During the same period, ILI was detected in 30Á9% of infants <6 months of age, with an even spread of influenza A, influenza B and RSV. 27 The influenza burden in children in a rural Indian community was found to be substantial with 11Á6% of ILI cases being caused by influenza A or B viruses. 28 These findings underscore the importance of maternal immunisation. 29 Transmission patterns Sequence analysis of influenza viruses isolated from students on a Singapore University Campus provided insights into the chain of transmission, showing that 62% of 32 viruses were highly similar, demonstrating that the majority of transmission was occurring on the university campus rather than from infections outside. 30 The effectiveness of surgical masks, hand hygiene and health education investigated in households in Hong Kong and Bangkok detected no significant difference in attack rate in cohorts using one of these interventions. 31 Further analysis of the data enabled some insights into the relative importance of aerosol, large droplet and contact transmission within the households. For influenza A infections, aerosol transmission appeared to be the most common route, whereas contact transmission caused the highest number of influenza B infections. 31 Use and effectiveness Neuraminidase inhibitors are commonly used for the treatment of influenza in Japan, typically following a positive result from a point-of-care (POC) test. During the 2009-2010 pandemic, over 20 million POC test kits were shipped to hospitals and clinics in Japan to enable rapid diagnosis, and 89% of treated cases were administered NAIs within 48 hours of symptom onset. 32 In Japan during 2013, oseltamivir and laninamivir each represented 40% of NAIs used, while zanamivir (15%) and peramivir (5%) use was considerably less. NAI effectiveness has been assessed in numerous observational studies in Japan. Oseltamivir effectiveness is significantly reduced in patients with delayed treatment, and duration of fever and viral shedding is longer in treated patients with influenza B compared to influenza A virus infections. 33, 34 The reduced effectiveness against influenza B viruses was also observed in zanamivir 33, 35 and laninamivir 35 trials. To determine whether NAIs reduced mortality during the 2009-2010 pandemic, data were compiled on 29 234 patients hospitalised with confirmed A(H1N1)pdm09 infection. 36 Compared with no treatment, NAI treatment was associated with significantly reduced mortality, with early treatment also showing a reduced risk of mortality compared to late treatment. Although there was no significant clinical effect when comparing late treatment with no treatment in hospitalised patients, there was a significant benefit in treating patients who arrive late into intensive care units. 36 In a household prophylaxis study, inhaled laninamivir given for either 2 or 3 days reduced the illness rate within households to 3Á9% and 3Á7%, respectively, compared to 16Á9% in households given a placebo. 37 A ferret model of oseltamivir prophylaxis has shown that while morbidity was significantly reduced, the prophylaxis regimes did not prevent infection nor significantly reduce virus load. 38 Resistance Although all four NAIs are sialic acid analogues, they have subtle differences in chemical structure and binding properties. Consequently, resistance patterns vary across NAIs. The most commonly detected NA substitution causing NAI resistance in N1-containing influenza viruses is H275Y, which confers resistance to oseltamivir and peramivir, but not to zanamivir and laninamivir. This resistance mutation became fixed in seasonal H1N1 viruses circulating in 2008-2009. 39 A late 2013 cluster of H1N1pdm09 viruses containing the H275Y substitution was detected in 38 (39%) of 97 H1N1pdm09 viruses from community patients not receiving NAIs in Sapporo, Japan, 40 reminiscent of a similar cluster of oseltamivir-resistant H1N1pdm09 viruses in community patients in Australia in 2011. 41, 42 Importantly, both sets of viruses contained permissive NA mutations (V241I and N369K) that have been shown in ferret studies to offset the destabilising and negative effect of the H275Y NA mutation. 43 In hospitalised influenza patients being treated with intravenous zanamivir, next-generation sequencing has been utilised to identify minor resistant virus populations. A total of five NA substitutions were identified in different viruses, including E119K and E119D; however, all apart from E119D were present in such low proportions that they could not be detected by Sanger 'population' sequencing methods. 44 The effects of various mutations in catalytic and framework residues of influenza B NA were investigated using reverse genetics and a range of functional assays. Four substitutions (D198E, I222T, H274Y and N294S) conferred reduced susceptibility to oseltamivir, while three substitutions (E119A, D198Y and R371K) caused highly reduced inhibition by oseltamivir, zanamivir and peramivir. 45 Two of these variants (H274Y, E119A) had in vitro replication fitness comparable to the NAI-susceptible viruses. To date, these substitutions have only been detected on rare occasions in circulating influenza viruses. An intravenous formulation of zanamivir showed both virological and clinical effectiveness without safety concerns in patients hospitalised with influenza in Japan. 46 A range of new adamantane derivatives have good antiviral activity in vitro and in animal models against H1N1pdm09 and H3N2 viruses that contain the S31N M2 ion channel substitution that confers resistance to amantadine. 47 Favipiravir is a novel pyrazinamide molecule that inhibits replication of various RNA viruses, including influenza types A, B and C (including oseltamivir-resistant strains), and has recently been licensed in Japan for the control of novel or reemerging influenza viruses. Its triphosphate metabolite is an RNA polymerase inhibitor which disrupts virus genome replication; synergy with oseltamivir has been demonstrated in pre-clinical models. 48 A phase II study in the US has shown that a twice-daily regimen decreased the titre and time to cessation of virus shedding, and had a significant benefit in reducing clinical symptoms (NCT01068912; www.clinicaltrials.gov). Subsequent phase III studies are currently ongoing (NCT02008344 and NCT02026349). A neutralising monoclonal antibody (MHAA4549A) which binds to the HA stalk of influenza A viruses in both group 1 and group 2 HA subtypes has been effective when given up to 72 hours post-infection in mice and ferrets infected with H5N1. 49 Phase I and IIa trials in humans showed that the antibody was well tolerated, had a mean half-life of 21Á9 days and was effective as therapy at high doses in experimentally infected volunteers (NCT01877785). Upcoming placebo-controlled phase IIb trials will target hospitalised influenza patients requiring oxygen and compare the combination of the monoclonal antibody with oseltamivir to oseltamivir monotherapy (NCT01980966). Other broadly neutralising antibodies against multiple clades of H5N1 have been generated by glycan masking of key HA antigenic residues to direct antibody responses to the more conserved stem region of the HA. 50 FluPep, a novel peptide that prevents virus entry into cells, has been shown in mouse studies to be effective in reducing virus titres in lungs, inflammatory cytokines and mortality. 51 Fludase (DAS-181) is a host-targeted therapeutic agent that removes sialic acid from cellular receptors in the respiratory tract, thus preventing influenza virus binding. Delivered topically, it is effective in animal models of lethal H5N1 and H7N9 infection, including a NAI-resistant R292K H7N9 variant. 52 In a phase 2 RCT, inhaled DAS181 reduced pharyngeal viral replication in uncomplicated influenza but did not reduce nasal virus loads or improve clinical outcomes. 53 Another receptor-targeted approach is the development of multivalent sialic acid-binding proteins; 54 a single administration 7 days pre-infection resulted in the protection of 80-100% of mice from lethal H7N9 challenge. 55 Apart from blocking sialic acid, the compound appears to stimulate the expression of pro-inflammatory mediators, thereby 'preparing' the immune system for subsequent influenza infection. When delivered 24 hours post-infection, protection was, however, only 20-40%. 55 Although drug resistance is considered less likely to occur with host-directed therapies, escape mutants have developed rapidly following exposure to a host-directed vacuolar ATPase-inhibiting drug. 56 Furthermore, following serial passage of different viruses in the presence of bafilomycin A1, two HA mutations were selected (A19T and S210N) which resulted in reduced drug susceptibility and increased virulence in mice. 56 Multiple influenza vaccine effectiveness (IVE) studies have used the control test negative design approach to estimate IVE during early and late phases of influenza seasons, the 2009 pandemic, and by age or target groups. Typically, IVE estimates range from 40% to 60% each season. 57 Future studies will investigate IVE with respect to the type of influenza vaccine used, whether IVE differs between the start and end of the season and the effect of previous vaccination. In Japan in 2013/14, IVE for influenza A in children aged 1-5 years averaged 72% (95% CI 64-79), dropped to 48% (95% CI 31-61) in children aged 6-12 years and was not apparent against influenza B in any age group (À1%, 95% CI À19 to 14). 58 Influenza vaccine effectiveness is known to be lower in adults over 65 years of age, a group that accounts for >60% of seasonal influenza-related hospitalisations and >90% of influenza-related deaths. In an effort to improve IVE in the elderly, recent RCTs have investigated the use of adjuvants, intradermal injection and higher doses of antigen. While the use of AS03-adjuvanted influenza vaccine was only moderately superior to non-adjuvanted vaccine in the elderly, 59 the use of a high-dose vaccine containing four times the standard level of HA (60 lg per virus) did result in improved effectiveness compared to the standard dose vaccine. 60 For vaccine manufacturers, generating high-growth reassortants of certain circulating viruses can be challenging. A recent study used random mutagenesis of PR8 and selection of high-growth clones in MDCK and Vero cells to derive a high-growth version of PR8. 61 Reassortment of the highgrowth PR8 virus with the HA/NA of either H5N1, H7N9 or seasonal influenza viruses showed that yields significantly exceeded equivalent reassortants that contained the internal genes of the 'normal' PR8 virus. 61 As of July 2014, the number of confirmed cases of MERS-CoV has exceeded 830, with at least 288 associated deaths. 62 The majority of cases have involved patients with comorbidities (76%) and are predominately males (63%) with a median age of 47. 63, 64 Fewer than 25% of patients have reported contact with animals including dromedary camels, which have been shown to be one likely animal reservoir based on sero-positivity and detection of MERS-CoV. 65 More than 25% of the infections have been in healthcare workers, and the large number of nosocomial infections is likely due to inadequate infection control in hospitals plus enhanced surveillance that has detected a substantial number of mild or asymptomatic infections. 63 Outside hospital, the burden of disease is likely to be larger than has been reported. 66 Serological analysis of several UK patients found a rapid rise in antibodies from day 10, and that titres were maintained for at least 300 days post-infection. Anti-S (spike glycoprotein) antibodies are responsible for virus neutralisation. Importantly for serological analyses, patients who experience only mild disease may mount only a modest serological response. 67 Sequential samples from three cases involved in a chain of transmission were extensively analysed using next-generation sequencing. 68 Various minority variants were detected, of which some were transient while others were transmitted, and there was evidence of variation in frequency of some variants in different body compartments. Various therapeutic options have been investigated for the treatment of MERS-CoV, but no therapy of proven value currently exists. The use of SC was associated with adverse outcome in SARS 69 and is not recommended for MERS-CoV. Many agents have shown inhibitory effects against MERS-CoV in cell culture including interferon +/À ribavirin, cyclosporine A, mycophenolic acid, chloroquine and lopinavir. 70 Interferons, lopinavir, mycophenolate, possibly alisporivir and combinations are reasonable choices for testing in controlled clinical trials. Exploratory post hoc metaanalysis of studies related to SARS and severe influenza has shown a significant reduction in mortality following convalescent plasma treatment compared to placebo or no therapy (odds ratio 0Á25; 95% CI 0Á14-0Á45). 71 Thus, the early use of virus-specific neutralising antibodies in the form of convalescent plasma and monoclonal or polyclonal neutralising antibodies for treatment of MERS-CoV has the highest likelihood of clinical benefit. 64 Modalities with risks likely to exceed benefits include SC, ribavirin monotherapy and IVIG. 72 Respiratory syncytial virus (RSV) Respiratory syncytial virus disproportionately impacts children in low-income countries. 73 Almost all children will have been infected with RSV by their 2nd birthday, and it is the number one cause of hospitalisation of infants in the US, causing 10 times more infant deaths than influenza. 74 In addition, RSV infects 3-10% of adults annually and accounts for 5-15% of community acquired pneumonia (CAP) and 9-10% of hospitalisations, 75 a burden of disease that approaches that caused by influenza. In a study, conducted in Hong Kong, of 607 hospitalised adults with RSV, 40% had pneumonia and 70% required supplementary oxygen; mortality rates and duration of hospital stay were similar to those observed for influenza patients. 75 Approximately, 15% of hospitalised RSV patients had bacterial superinfections. Although corticosteroids were used to treat 38% of patients, treatment had no benefit on clinical outcome, and instead increased bacterial secondary infections and caused a longer duration of illness. 75 RSV replication appears prolonged in patients with comorbidities and LRT complications. Palivizumab prophylaxis of premature infants of <6 months of age has been shown to reduce hospitalisation due to RSV by 55%. 76 Preventing RSV during infancy has been associated with reduction of wheezing later in life. 77 Trials of other monoclonal antibodies have typically shown that they do not achieve superiority compared to palivizumab and therefore do achieve licensure. Furthermore, treatment with neutralising monoclonal antibodies does not appear to reduce virus load or disease severity in hospitalised infants. 78 Alternative options for RSV therapy to be assessed in future clinical trials include inhaled nanobodies, aerosolised peptides, nucleoside analogues and RNA-interference molecules. Human rhinoviruses (HRV) usually cause mild acute respiratory infections, but on occasions can also cause more severe respiratory infections, including exacerbations of asthma and COPD. Of 115 Japanese children with asthma, a respiratory virus was detected in 86%, of which HRV (n = 36) or RSV (n = 47) were most common. 79 Ex vivo bronchial epithelial cells from people with asthma are more susceptible to HRV infection, due to deficient induction of IFN-b and IFN-lambda. In a study of 147 asthmatics on inhaled corticosteroid therapy, with a history of virusassociated exacerbations, patients were randomised to 14-day treatment with inhaled IFN-b or placebo within 24 hours of developing cold symptoms. Patients who received IFN-b had enhanced morning peak expiratory flow recovery, reduced need for additional treatment and boosted innate immunity as assessed by blood and sputum biomarkers. In an exploratory analysis of a subset of more difficult-to-treat asthma (n = 27 IFN-b; n = 31 placebo), worsening of symptoms increased significantly in the placebo group, but was prevented by IFN-b (P = 0Á004). 80 A picornavirus-specific antiviral, vapendavir, was found to reduce symptom scores, lower bronchodilator puffer use and reduce viral load in asthma patients with an URTI due to HRV. 81 Diagnostics Point-of-care tests that can deliver a result in 15 minutes have been available in many countries for the last decade, but while having good specificity, the sensitivity has typically been poor, ranging from 10% to 80% compared to PCR or culture. Their use in emergency departments of hospitals can result in reduced unnecessary antibiotic use and an increased likelihood of discharge. Newer immunofluorescence-based POC tests with improved sensitivity are being developed. In addition, the Quidel Sofia POC test may be linked via the internet such that results can be reported in real-time to central databases. Other POC tests are using photographic silver amplification immunochromatography technology to increase sensitivity. 82 PCR remains the gold standard for virus diagnostics with an ability to be rapid, sensitive, specific and to identify a wide range of pathogens via different assays. The ability to multiplex multiple pathogen targets allows costs to be reduced in a diagnostic setting. New closed-system technologies which involve only minimal hands-on time (a few minutes) and that conduct both automated nucleic acid extraction and PCR for multiple pathogens are now available, but are currently limited for clinical diagnostic purposes due to low-throughput capabilities. 83 Next-generation sequencing technologies and PCR-based analyses with increasing sensitivity both offer considerable scope in diagnosis, although our current understanding of the clinical impact of pathogens at low levels or the presence of variants as minor virus populations is limited. Providing low-cost, sensitive assays for diagnosing respiratory virus infections in low/middle-income countries is challenging, but has the potential to improve treatment and avoid unnecessary antibiotic use in these regions. Repurposed drugs for respiratory viral infections Nitazoxanide (NTX) is an antiparasitic agent approved for Giardia and Cryptosporidium infections that also inhibits replication in vitro of influenza and other respiratory viruses. 84 Treatment with NTX 600 mg twice daily for 5 days was associated with a reduction in the duration of symptoms in participants with acute uncomplicated influenza. 85 In a subset analysis of 238 patients with no confirmed virus infection, treatment with NTX 600 mg also led to a shorter time to alleviation of symptoms in comparison to placebo (88Á4 versus 105Á7 hours, P = 0Á02). 86 Systemic corticosteroids for respiratory virus infections A review of prospective observational studies has shown that SC increased the risks of mortality and morbidity (e.g. secondary infections, hospital-acquired pneumonia) in severe infection due to influenza A(H1N1)pdm09 especially with delayed antiviral therapy. 87 During SARS infections, a higher risk of avascular necrosis and prolonged virus shedding were observed in patients who had received highdose SC therapy. 88 It is therefore important to avoid the use of high-dose SC in severe respiratory viral infections outside the context of clinical trials. Larger trials are needed to resolve the uncertainty regarding the effect of early SC therapy in ARDS. Low-dose SC is indicated for management of refractory septic shock, 89 and a short course of SC is indicated for acute exacerbations of obstructive airway diseases (asthma, COPD) 90 Current evidence does not support a clinically relevant effect of systemic or inhaled glucocorticoids on admission or length of hospitalisation for acute viral bronchiolitis in infants and young children. 91 Respiratory syncytial virus, influenza viruses, parainfluenza (PIV) viruses and adenoviruses (AdVs) cause the most serious disease in immunocompromised hosts, but other respiratory viruses are becoming increasingly appreciated as a cause of both upper and lower respiratory tract disease. The potential for these viruses to cause lower respiratory tract infections (LRTI) after transplantation varies. Human metapneumovirus infections have similar outcomes to RSV infection in hematopoietic stem cell transplant (HSCT) recipients, including potentially severe and fatal pneumonia. HRV and coronavirus infections are very frequent in transplant recipients, but severe lower respiratory tract disease is uncommon. In a prospective study of 112 lung transplant recipients, the virus infection rates upon screening, routine and emergency visits were 14%, 15% and 34%, respectively. Picornaviruses were identified most frequently in nasopharyngeal (85/140; 61%) and BAL specimens (20/34; 59%). Asymptomatic virus carriage, mainly of picornaviruses, was found at 10% of screening visits. Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels. The hospitalisation rate was 50% for influenza and PIV and 16Á9% for other viruses. Acute rejection was not associated with virus infection. 92 The risk factors for severe LRTI among transplant recipients include early onset post-transplant (<3 m), steroid boluses, young children (<1 year), chronic GVHD, lymphopenia/lymphodepletion and allogeneic HSCT patients. 92 Influenza Immunocompromised patients with influenza exhibit more complications, longer virus shedding and more antiviral resistance, while often demonstrating milder clinical symptoms and signs on initial clinical assessment. 93 Influenza A (H1N1)pdm09 viruses have the potential for rapid emergence of oseltamivir resistance and causing severe morbidity, particularly in immunocompromised patients with lymphopenia and delayed antiviral therapy. 94 Influenza viraemia may serve as a marker for overall poor outcome with increased risk of progression to LRTI, hypoxaemia, respiratory failure and death. Influenza RNA in blood (viraemia) was detected in nine of 79 (11Á4%) HSCT recipients with influenza. Among patients with LRTI, viraemia was associated with increased hazards of overall as well as influenzaassociated death (hazard ratio 3Á5, 1Á1-12). 95 In 143 HSCT recipients with documented seasonal influenza infection, treatment with high-dose corticosteroids was associated with a trend towards prolonged virus shedding [(OR), 3Á3; 95% CI 1Á0-11; P = 0Á05], whereas antiviral therapy initiated to treat upper respiratory tract infection (URTI) was associated with fewer cases of LRTI (OR, 0Á04; 95% CI, 0-0Á2; P < 0Á01) and fewer hypoxaemia episodes (OR, 0Á3; 95% CI, 0Á1-0Á9; P = 0Á03). 96 In view of the risks of prolonged replication and drug resistance emergence, 97 a longer duration and a higher NAI dose may be beneficial. Early therapy was consistently demonstrated to have improved outcomes. [98] [99] [100] [101] Other treatment options under study are the use of triple combination therapy with amantadine, oseltamivir and ribavirin, 102 or intravenous peramivir 103 or zanamivir. 104 Therapy of influenza in lung transplant recipients is associated with a reduced risk of developing bronchiolitis obliterans syndrome. 105 Parainfluenza DAS181 is inhibitory for PIV and influenza viruses, including those resistant to the amantadine and NAIs 106 and may be effective in treating immunocompromised patients with severe PIV lung disease. 107 In a study of four severely immunocompromised children with PIV disease treatment with DAS181 for 5-10 days, by dry powder inhalation or nebulisation, was well tolerated. Transient increase in serum alkaline phosphatase, liver function and coagulation tests were observed, but nasal wash virus loads were reduced in all patients within 1 week with improved clinical features. 108 In a RCT of lung transplant recipients with RSV infection, the incidence of new or progressive bronchiolitis obliterans syndrome at day 90 was significantly reduced in 16 patients who received a small interfering RNA against the RSV Ngene (ALN-RSV01) compared with placebo (n = 8) (6Á3% versus 50%, P = 0Á027). 109 In a larger follow-up multicentre phase IIb study, treatment with ALN-RSV01 showed a greater than eightfold reduced risk in developing bronchiolitis obliterans syndrome at day 180. 110 Adenovirus is a serious, often fatal infection in immunocompromised patients, especially in HSCT recipients. The control of AdV is mostly T-cell mediated, and therefore, patients who have received T-cell suppressive regimens are at an increased risk for AdV infection. The annual incidence of AdV infections in HSCT recipients ranges from 5% to 50%, and is increasing, likely due to increased use of T-celldepleted allografts and cord blood as source. The mortality rate is up to 80%. 111 Brincidofovir (BCV; formerly CMX-001) is an orally bioavailable lipid-conjugate of cidofovir (CDV) that provides high intracellular concentrations of CDV diphosphate with a long intracellular half-life (up to 4-6Á5 days). BCV is 65-fold more potent against AdV than CDV in vitro with a low risk of myeloidor nephrotoxicity, but gastrointestinal side effects are more common. 112 In a retrospective study of 13 immunocompromised patients given BCV for AdV disease after failing or intolerance to i.v. cidofovire nine patients (69Á2%) demonstrated a virological response (VR), which was defined as a 99% drop from baseline or undetectable AdV DNA in serum by week 8. Patients with VR had longer survival than those without VR (median 196 days versus 54Á5 days; P = 0Á04). 113	How many deaths were associated with MERS-CoV as of July 2014?	5,321	288	21,748
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What illness is caused by the 2019-nCOV Coronavirus?	1,149	The novel coronavirus (2019-nCoV) infection caused pneumonia.	563
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	In addition to oral swabs, which tests detected the presence of 2019-nCOV virus?	1,151	the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients).	782
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What is the relationship between the presence of virus in blood and anal swabs and disease severity?	1,160	all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage.	906
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	Which patients were classified as severe in Chinese guidelines?	1,168	Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included.	4,705
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What is the relationship between the presence of virus in blood sample and disease severity?	1,169	In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases.	5,150
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What test could give an indication for special care for 2019-nCOV patients?	1,171	presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care	9,149
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What is the relationship between the presence of virus in anal swabs and disease severity in 2019-nCOV?	1,170	In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases	5,505
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What could be the implication of 2019-nCOV virus in anal swabs?	1,172	digestive tract might be one extrapulmonary site for virus replication	10,737
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What could account for the high transmission rate of the 2019-nCOV virus?	1,173	Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus.	11,258
2,519	Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.	What could account for the dissemination of the 2019-nCOV virus across the whole body?	1,174	We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa	11,770
1,618	‘Tiny Iceland’ preparing for Ebola in a globalized world https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507955/ SHA: efd94d1135c5ee11c2af624b344881e079a5ce7a Authors: Gunnlaugsson, Geir; Hauksdóttir, Íris Eva; Bygbjerg, Ib Christian; Pinkowski Tersbøl, Britt Date: 2019-05-07 DOI: 10.1080/16549716.2019.1597451 License: cc-by Abstract: Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: "How will I manage working in this for hours?" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark.	When did the World Health Organization declare the Ebola epidemic in West Africa as a Public Health Emergency of International Concern?	2,178	8 August 2014	2,494
1,618	‘Tiny Iceland’ preparing for Ebola in a globalized world https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507955/ SHA: efd94d1135c5ee11c2af624b344881e079a5ce7a Authors: Gunnlaugsson, Geir; Hauksdóttir, Íris Eva; Bygbjerg, Ib Christian; Pinkowski Tersbøl, Britt Date: 2019-05-07 DOI: 10.1080/16549716.2019.1597451 License: cc-by Abstract: Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: "How will I manage working in this for hours?" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark.	What is PPE?	2,181	Personal Protective Equipment	6,407
1,618	‘Tiny Iceland’ preparing for Ebola in a globalized world https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507955/ SHA: efd94d1135c5ee11c2af624b344881e079a5ce7a Authors: Gunnlaugsson, Geir; Hauksdóttir, Íris Eva; Bygbjerg, Ib Christian; Pinkowski Tersbøl, Britt Date: 2019-05-07 DOI: 10.1080/16549716.2019.1597451 License: cc-by Abstract: Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: "How will I manage working in this for hours?" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark.	Where did the 2014 Ebola epidemic in West Africa spread to?	2,183	neighbouring sub-Saharan countries, North America, and Europe	20,518
1,618	‘Tiny Iceland’ preparing for Ebola in a globalized world https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507955/ SHA: efd94d1135c5ee11c2af624b344881e079a5ce7a Authors: Gunnlaugsson, Geir; Hauksdóttir, Íris Eva; Bygbjerg, Ib Christian; Pinkowski Tersbøl, Britt Date: 2019-05-07 DOI: 10.1080/16549716.2019.1597451 License: cc-by Abstract: Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern (PHEIC) under the International Health Regulations (IHR) [1] . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems [2] [3] [4] . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola [5] , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines [6] , flights were cancelled and cross-border movement curtailed [7] . The epidemic caused public concern outside West Africa [8] , as fear and racism found fertile ground [9] [10] [11] , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases [1] . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' [12] . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied [13] . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A (H1N1) pandemic revealed that the way plans were framed varied considerably, and '[told] us something about how the different countries want pandemics and preparedness to be understood by the public' [14] . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health (DH) first reported on the epidemic on 8 April 2014 [15] . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola [16] [17] [18] , despite its ranking as one of the best in the world [16] . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola [19] . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital (LSH), the Department of Civil Protection and Emergency Management (DCPEM), and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control (ECDC) and WHO, in addition to Nordic collaborators in epidemic preparedness [20] . At the same time, it was regarded as highly unlikely that Ebola Virus Disease (EVD) would spread in the country [21] . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities [22] [23] [24] [25] . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment (PPE) entered the plane, but quickly ruled out Ebola [26] . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland [16, 27] . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness [28, 29] . Staff in managerial positions were contacted by one of the authors (GG) for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team (ETT), the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors (ÍEH), a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room (ER). They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively (see supplementary material). The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee (VSN- and Landspitali University Hospital (LSH 13-16, 4 February 2016) . Reporting on the results was guided by the COREC guidelines [30] ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH [31] , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD [31] . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as [we] could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness [would have been] needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: "How will I manage working in this for hours?" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these [non-clinical] people belonged in the team, because this is a very clinical environment in addition to having to be in this costume [PPE] with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, [we] help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew (s)he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe [32] , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic [33] , in the EU region [34, 35] , and the US [36] . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' (theme 1) refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis [37] emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' [38] . The West African Ebola epidemic was covered widely by media [39] , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers [40] , in contrast to the ongoing epidemic in the Democratic Republic of Congo [41, 42] . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges [35] . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere [43] . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation [44] . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics [35] . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada [45, 46] . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies (those of health professionals), clinical architectures (e.g. isolation wards), and technical artefacts (gloves, protective suits, disinfectants, etc.) [47] . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland [48] . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' (things always have a way of working out in the end). The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced [38] . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 [35] . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 [49] . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere [44] , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics [38] . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola (or other contagious global health threats) as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer (CMO) for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland (number VSI- ). Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 (number LSH [13] [14] [15] [16] . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark.	What are the prerequisites for successful emergency preparedness for an epidemic?	2,184	connectedness between health professionals, technological devices, and knowledge	25,212
1,722	Recent Progress in Studies of Arterivirus- and Coronavirus-Host Interactions https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397358/ SHA: f5e974ef3a8c983ae63ac4f4aa2b6ec0e3678032 Authors: Zhong, Yanxin; Tan, Yong Wah; Liu, Ding Xiang Date: 2012-06-19 DOI: 10.3390/v4060980 License: cc-by Abstract: Animal coronaviruses, such as infectious bronchitis virus (IBV), and arteriviruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), are able to manifest highly contagious infections in their specific native hosts, thereby arising in critical economic damage to animal industries. This review discusses recent progress in studies of virus-host interactions during animal and human coronavirus and arterivirus infections, with emphasis on IBV-host cell interactions. These interactions may be directly involved in viral replication or lead to the alteration of certain signaling pathways, such as cell stress response and innate immunity, to facilitate viral replication and pathogenesis. Text: Coronaviruses, together with arteriviruses and toroviruses, belong in the order Nidovirales, a group of large, non-segmented, positive sense and single stranded RNA animal viruses that produce an extensive 3'-nested set of subgenomic mRNAs for transcription during infection [1] (Table 1) . Nidoviruses such as avian infectious bronchitis coronavirus (IBV), human coronavirus 229E (HCoV-229E), equine arteritis virus (EAV) and the porcine reproductive and respiratory syndrome arterivirus (PRRSV) are important pathogens of both human and animals [2] [3] [4] , and are commonly associated with mild respiratory and enteric diseases, although they are also known to cause more OPEN ACCESS critical lower respiratory tract illness, such as the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic that occurred in 2003 [5] . As these viruses infect livestock, coronaviral and arteriviral infections in farms have resulted in large-scale economic losses in farming nations, and are therefore of exceptional veterinary research value. Coronaviruses in fowls, as exemplified by the highly contagious IBV in chickens, can be highly lethal to young chicks. IBV is the etiological agent of infectious bronchitis, an avian disease that is mainly associated with upper respiratory and urogenital tract infections in adult chickens, and to a lesser extent, nephrogenic infections, or inflammation of the kidneys [6, 7] . The impact of IBV infection is also emphatically increased as a consequence of its enhancement of diseases associated with lethal co-infections by bacteria and mycoplasmas [8, 9] . Domestic animals such as cats and dogs are also susceptible to coronaviruses. Feline coronaviruses, especially feline infectious peritonitis virus (FIPV)-a mutation of Feline Enteric Coronavirus (FECV), may induce lethal diseases in cats [10] , while canine coronavirus (CCoV) infections, which cause canine enteric illness in dogs, are prevalent as well [11] . In larger livestock like pigs and cattle, on the other hand, coronaviruses and arteriviruses typically establish enteric infections. An infection or outbreak can cause severe economic losses from the death of young offspring, lifelong impact on the yield of animal produce such as eggs and milk, weight losses and the general health of the population. Bovine coronavirus (BCV), for example, causes Winter Dysentery (WD) in adult cows and diarrhea in young calves [12] . The pathogenicity of these viruses is typically species-dependent, as is the severity of infection; they infect mainly their natural hosts and/or species that are closely related. Certain virus infections, however, can cross the species barrier, with the prime example being the zoonotic SARS-CoV, a novel coronavirus that is thought to have originated from bats before it adapted to its intermediate host, civet cats, and finally to humans [13] . Bat colonies, which are scattered worldwide, are widely known to play host to a variety of coronaviral and adenoviral pathogens while acting as natural wildlife reservoirs of these viruses [14] [15] [16] . Coronavirus infections are also generally tissue-specific-the Transmissible Gastroenteritis Coronavirus (TGEV), for example, affects mainly the gastrointestinal tract [17] that may lead to the onset of fatal watery diarrhoea and severe dehydration in pigs [18] , while human coronaviruses mostly cause respiratory infections [4] . With respect to their significance to the economy, vaccines have also been developed for many of these viruses in a bid to prevent localized infections from progressing into serious outbreaks. This has, however, proven to be a hard battle as the vaccines are unable to provide complete cross-protection among the various serotypes of each virus [19] . During infection, the virus replicates in the host cytosol amidst a myriad of host signaling pathways and systems such that interaction between the virus and the host systems is inevitable. Virus infection and the consequent host cell response also involve complicated interaction between various host cellular and viral networks. Virus-host interplay occurs at multiple points during the virus replication cycle, from entry to exit. The nature of such interactions can range from a simple exploitation of existing host machinery to destructive interactions that modulate the host environment to the advantage of the virus while inhibiting host activities. One of the most important interactions between virus and host is the modulation of host cell environment, such that the latter is converted into one in which the virus can replicate successfully. Viruses also regulate the differential expression of host genes, as well as various host antiviral defense mechanisms, for more efficient replication. Previous studies on the infection of different hosts by nidoviruses, for example, have shown various modifications in host innate immune and stress responses, cell cycle, autophagy and cell death pathways [20] [21] [22] [23] , all of which will be discussed in this review. The significance of host components being used to supplement the gene-poor virus in various processes cannot be dismissed, for although they typically serve as enhancers, they could also become major pathogenicity factors. A number of stimuli can precipitate apoptotic events, including cell homeostatic imbalance such as cell stress, and the binding of ligands to cell surface "death" receptors; these in turn trigger the onset of major apoptotic pathways: the extrinsic or intrinsic pathway [24] . The extrinsic pathway can be induced by several cytokine "death" receptors from the tumor necrosis factor (TNF) family, such as Fas (Apo1/CD95) [25] . Upon recruitment of their respective ligands, they form complexes that subsequently bind death effector domain (DED)-containing pro-Cysteine Aspartyl-Specific Proteases (pro-caspases), in particular pro-caspase-8, where the activation and consequent oligomerization of which further serves is a signal for downstream activations, thus pledging the doomed cell towards its own death [26] . The intrinsic pathway, on the other hand, is activated by the release of cytochrome c from the mitochondria into the cytoplasm [27] . In the cytosol, cytochrome c binds the apoptotic protease-activating factor (Apaf1); together, they form an apoptosome that leads to the release of active caspase 9. Apoptotic mitochondrial events are also regulated primarily through the activation of pro-survival and pro-apoptotic proteins [28] . The Bcl-2 family of proteins constitutes a critical control point in the regulation of apoptosis. They form three major protein subgroups: the Bcl-2 homology (BH) 3, or BH3-only proteins [e.g., BH3-interacting domain death agonist (Bid), BCL2-associated agonist of cell death (Bad)], Bax-like proteins [e.g., BCL-2-antagonist/killer 1 (Bak), BCL-2-associated X (Bax)] and the Bcl-2-like factors [e.g., Myeloid cell leukemia-1 (Mcl-1), Bcl-extra large (Bcl-X L )] [29] . BH3-only and Bax-like proteins are essential initiators of apoptosis while the Bcl-2-like proteins are pro-survival factors that safeguard the cells against apoptosis. Both caspase 8, from the extrinsic pathway, and caspase 9, from the intrinsic pathway, have been observed to activate the main effector caspase 3, which in turn activates a caspase cascade to eventually evoke the morphological hallmarks of apoptosis such as DNA fragmentation [30, 31] . A third apoptotic pathway, induced by prolonged endoplasmic reticulum (ER) stress, has also been shown to activate multiple downstream apoptotic targets, including rodent caspase 12, growth arrest and DNA damage-inducible gene 153 (GADD153), also known as the transcription factor C/EBP homologous protein (CHOP) as well as activation of the pro-apoptotic c-Jun NH2-terminal kinase (JNK) [32] . Human pro-inflammatory caspase 4, a nearly identical paralogue of the rodent form of caspase 12, has also been shown to possess comparable roles in ER-stressed apoptosis [33] . JNK activation is mediated by ER transmembrane protein kinases, while CHOP is triggered by ER stress at the transcriptional level [32] . The downstream apoptotic activities of both JNK and CHOP have also been postulated, at least in part, to be connected with the Bcl-2 family of proteins (Bak and Bax) for recruitment to the ER and subsequent initiation of apoptosis in response to stress [32] . As viruses depend on the host cells they infect in order to reproduce, apoptosis is often employed as an important host antiviral defense mechanism that, as a protective measure, leads to the abortion of virus infection such that viral productivity and persistent infectivity is consequently limited [34] . In many cases, p53 and the Bcl-2 family of proteins have been shown to be the main mediators that induce the beleaguered cell to undergo self-induced death at various stages of the infection cycle [35] . Moreover, host endosomal membranes are forced to undergo conformational changes for the fusion of virus and host cell membranes during virus uncoating; membrane integrity is also antagonized during the process of virus disassembly. As such, these drastic alterations to membranes may elicit downstream pro-death signals that prompt infected cells to commit suicide [36] . However, certain viruses have evolved strategies to both counteract and induce apoptosis in order to maximize the production of virus progeny and promote its spread to neighbouring cells. An increasing number of known viruses from different families, including arteriviruses, have been found to induce apoptosis during their infection cycle, which may possibly contribute to the cytotoxicity associated with virus infections, especially during late stages of infection [37] . Membrane-bound cell fragments are also produced as apoptotic bodies to be phagocytosed by surrounding cells. This provides an excellent method for a virus to disperse its progeny without eliciting host immune response [38] . While more comprehensive work needs to be done to paint a clearer picture of how coronaviruses and arteriviruses regulate apoptosis during infection, recent reports have suggested the possible activation of more than one apoptotic pathway during infection. EAV, an arterivirus that is prevalent among global horse populations and which may induce abortions in pregnant mares [39] , has been shown to activate apoptosis through the initiation of caspase-8-dependent mechanisms, which is followed by mitochondria-dependent caspase-9 activation mechanisms [40] . PPRSV, a virus that causes respiratory tract problems in young pigs and has a commercially significant impact on swine industries worldwide as a result of reproductive impairment in breeding livestock [41] , has also been implicated to regulate both apoptosis and necrosis during infection [42, 43] . Replication of TGEV in porcine kidney cells, as well as that of canine coronavirus type II (CCoV-II) in canine fibrosarcoma cells, on the other hand, has been reported to induce apoptosis both through Fas/FasL-activation and mitochondrial-dependent pathways [44, 45] . Figure 1 . Viral genes and the activation of apoptosis. Extrinsic signals from receptors (e.g., Fas ligand) culminate in the activation of caspase 8, which activates the effector caspase 3 while intrinsic signaling requires the participation of the mitochondria in releasing cytochrome c (shown as circles labeled "C") to activate caspase 9 for the downstream activation of caspase 3. Key proteins in the intrinsic apoptosis signaling pathway are p53, both pro-apoptotic (e.g., Bax, Bak) and anti-apoptotic proteins (e.g., Bcl-2, Bcl-X L) from the Bcl-2 family. Viral genes (see yellow boxes), which act at multiple points along the different signaling pathways, target both the extrinsic and intrinsic apoptosis signaling pathways and enhances the pro-apoptotic effect brought upon by virus infection. Anti-apoptotic proteins (black oval) listed are key anti-apoptotic members from the Bcl-2 family of proteins. Other pathways triggered by coronavirus infections, such as ER stress and DNA damage response, activate apoptotic signaling pathways as well. Although IBV is an avian virus, it can acclimate to primate cells and has been demonstrated to conquer the host species barrier and become zoonotic to infect both human and animal cells [46, 47] . IBV also triggers apoptosis during the late stages of its cytolytic infection cycle. Specifically, IBV-induced apoptosis has been shown to involve Bcl-2 family of proteins [48] , through caspase-dependent [49] and p53-independent [50] pathways in cultured mammalian cells. The modulation of Bcl-2 family proteins during IBV infection has also been postulated to be under the regulation of signaling pathways such as ER stress and Mitogen-Activated Protein Kinase/Extracellular signal-Regulated Kinase (MAPK/ERK) pathways [48] . The effects of these pathways on the regulation of other host defenses will be discussed in later paragraphs. While viral genes can also manipulate the induction of apoptosis to the benefit of viruses, only two have been reported in the case of coronaviruses. The unique SARS-CoV encoded protein, 7a, was discovered to have caspase-dependent, pro-apoptotic functions and may function as a plausible source of virus-derived apoptotic signal [51] , while TGEV accessory gene 7, present only in coronaviruses classified under genus 1 [52] , thwarts virulence and host-induced antiviral mechanisms through the negative modulation of downstream caspase-dependent apoptotic pathways [53] (Figure 1 ). The maintenance of apoptosis is also important in the establishment and governance of immune responses in a cell. A loss in the control of apoptosis leads to an imbalance in cell homeostasis, which ultimately affects immune sensitivity [54] . The presence of apoptotic cells, particularly in existence with infectious agents, may also lead to the mobilization and initiation of innate immune defenses [55] . This crosstalk between apoptosis and innate immunity is therefore of considerable importance during pathogenic infection and can be manipulated by both host and pathogen, either as a form of immune defense or immune evasion, respectively [56] . When the host immune system is exposed to viral pathogens, it reacts straightaway by triggering a diverse array of defense mechanisms in order to establish a more efficacious shield. The first line of defense is the mounting of an innate immune response, as characterized by the increased production of type I interferons (IFN-α and IFN-β) and other inflammatory cytokines. These, in turn, choreograph the expression of downstream IFN-stimulated genes (ISGs) and activate several signaling pathways, all of which collaboratively lead to the induction of a protective antiviral state and, subsequently, the inhibition of both viral replication and proliferation [57] . The cytokine family of interferons is dedicated to the conveyance of the presence of infection, as well as the expedition of numerous connections among the cells that provide protection against, or eradication of, foreign pathogens. Other than interfering with viral progeny production in host cellshence the name 'interferon'-IFNs also induce Natural Killer (NK) cells and macrophages to 'kill' or engulf infected cells, increase antigen presentation to thymus (T) cell lymphocytes for rapid recognition of infected cells and bring about virus resistance to new uninfected cells [58] . IFNs are conventionally classified into three types: Type I (IFN-α, IFN-β and IFN-ω), Type II (IFN-γ) and the more recently identified Type III (IFN-λ1, IFN-λ2 and IFN-λ3) [59, 60] . Over time, mammalian hosts have gradually developed a multitude of cellular sensors for the detection of viral infection, and it is the involvement and operation of these cellular protein receptors that eventually leads, through an intricate network of pathways, to the expression of type 1 IFNs. Major receptor systems that conduct immune surveillance and trigger the production and subsequent release of type I IFNs are known as pattern recognition receptors (PPRs), which detect viral infection through the identification of various pathogen-associated molecular patterns (PAMPs); PPR families include the toll-like receptor (TLR), RIG-like helicase (RLH) and Nucleotide-binding oligomerization domain (NOD)-like receptor (NLRs) families [61] . The TLR family is mainly made up of transmembrane proteins, which conduct surveillance from the cell surface, as well as cellular compartments such as the endosome or ER, constantly scanning the extracellular environment for PAMPs that can be derived from a wide range of microorganisms, including viruses and bacteria. The expression of TLRs appear to be cell-specific and is mainly found in antigen-presenting cells such as dendritic cells (DCs), monocytes and macrophages, as well as on B cells [62] . TLRs recognize a wide variety of PAMPs, and the recognition of these ligands can be converted into specific intracellular responses through the direct interaction of the TLR toll-interleukin 1 receptor (TIR) domain with one of its cytoplasmic TIR-containing signaling adaptor molecules, such as myeloid differentiation primary response protein 88 (MyD88) and TIR domain-containing adaptorinducing IFNβ (TRIF) [63] . Viral recognition by TLRs has been detailed in several reports [64, 65] . In particular, the activation of TLR-3, -4, -7, -8, and 9 can also culminate in type I IFN production [65, 66] . TLR3, as a general viral sensor, detects mainly through double stranded RNA (dsRNA), a replication intermediate of both DNA and RNA viruses; TLR4 recognizes envelope proteins from viruses such as mouse mammary tumor virus (MMTV); TLR7 and TLR8 have been identified to recognize ssRNA viruses like influenza and vesicular stomatitis virus (VSV); TLR9 detects dsDNA viruses such as herpesviruses [63] . NLRs are stimulated by microbial agonists and collaborate with TLRs to evoke intracellular immune responses through MAPK and caspase signaling cascades upon sensing bacterial components [67] . NLRs are also known to sense both PAMPS from infectious agents and DAMPs (danger-associated molecular patterns) that arise as a result of insult or injury to the cell, as well as those derived from the environment [68] . Although the direct binding of virus-derived PAMPs to NLRs is yet to be reported, structural and functional studies of the C-terminal domain of NLRX1, a mitochondrial member of the NLR family, has highlighted its ability to bind both ssRNA and dsRNA, implying that some NLRs may be capable of binding viral RNA directly as well [69] . As such, the notion of NLR-mediated recognition of coronaviruses is, therefore, probable and could be further investigated. The RLH family of purely cytoplasmic PRRs is made up of the following: retinoic acid inducible gene-I (RIG-I or DDX58), melanoma differentiation-associated gene-5 (MDA5 or IFIH1), and laboratory of genetics and physiology 2 (LGP2). RIG-I and MDA5 are PRRs with two N-terminal caspase-recruitment domains (CARDs) followed by a DExD/H box RNA helicase domain; LGP2 lacks the signaling caspase recruitment domains but shares a helicase domain of similar homology and is thought to serve as a regulator of the former [70] . RIG-I and MDA-5 both sense cytoplasmic dsRNA, which the host recognizes as 'non-self', via the N-terminal CARDs [71] . However, the two PRRs each sense distinct PAMPs, depending on the length of viral dsRNA, from different RNA viruses. In addition to long dsRNAs (>2 kb) such as the synthetic dsRNA analogue poly-inosinic poly-cytidylic acid [poly(I:C)], MDA5 also recognizes picornaviruses and noroviruses [72, 73] . RIG-I, on the other hand, responds to paramyxoviruses, flaviviruses, orthomyxoviruses and rhabdoviruses [61, 74, 75] . This is through its recognition of a variety of ligands such as relatively short dsRNA (19-mer to 1 kb) and ssRNA (single stranded RNA), both preferably in the presence of a 5'-triphosphate end, full-length RNA viral genomes, the presence of secondary structures such as poly-uridine motifs within 5'-triphosphate genome termini or longer RNA sequences without 5'-triphosphates, such as 3' untranslated regions (UTRs) of the genome [76] [77] [78] [79] [80] . While the elicitation of TLRs and RLRs by PAMPs trigger their distinct signaling cascades through divergent downstream effectors at varying efficacies, they ultimately cross paths at the juncture of transcriptional activation of interferon regulatory factor 3 (IRF3) [81] , IRF7 [82] and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [83] [84] [85] [86] , all of which translocate to the nucleus and activate the transcription of both type I interferons (IFNα and IFNβ) and inflammatory cytokines that eventually culminates in the concerted induction and development of adaptive antiviral immune response. The expression and induction of interferons from the cells occurs in response to viral insults and tumor growth [87] . Type I interferons, the major group of cytokines in innate anti-virus defense, bind a specific cell surface heteromeric receptor, the interferon-α/β receptor (IFNAR), which composed of two subunits, IFNAR1 and IFNAR2 [88] . The best characterized type I IFNs can be classified into two major groups, the immediate-early genes (such as IFNβ) that are triggered by the initial response to virus infection, and the delayed-set (such as IFNα subtypes) that rely on a secondary de novo protein synthesis pathway [89] . IFN expression is regulated by IFN regulatory factors. In particular, IRF3 and IRF7 play vital roles in activating innate immune response through their respective antiviral response [66] . IRF3 has been functionally characterized to consist of a nuclear export signal (NES), a DNA-binding domain (DBD), a C-terminal IRF association domain (IAD), as well as a number of phosphorylation sites as well as two auto-inhibitory domains that prevents a constitutive activation of the NES, DBD and IAD [90] . Normally found in an inactive cytoplasmic form, IRF3 is phosphorylated as a consequence of virus infection. This activation signal exposes the DBD and IAD and results in the dimerization of IRF3, either as a homodimer or as a heterodimer with IRF7, allowing the activated IRF3 to form a complex with the transcriptional co-activator cAMP-response element-binding protein (CREBP) to translocate to the nucleus and bind to DNA to trigger the transcription and expression of immediate-early IFNs, which signals the JAK-STAT (Janus kinase-Signal Transducer and Activator of Transcription) pathway through the binding of the IFNAR [90] . This leads to the formation of a STAT1-STAT2 heterodimer that teams up with the interferon regulatory DNA binding factor IRF9, which together constitute an activated heterotrimeric factor, the interferon-stimulated gene transcription factor ISGF3, that, through the recognition and binding of specific ISREs, induces downstream expression of innate immunity genes for host defense against virus invasion [91] . In contrast to the constitutive expression of IRF3, IRF7 is involved in the positive feedback regulation of IFN production [89] . Expressed only in minute amounts, the induction of IRF7, via virus-induced activation of ISGF3, results in either homo-dimerization or hetero-dimerization with IRF3 and is subsequently followed by nuclear translocation for the activation of both IFN and IFN genes [92, 93] . Coronaviruses and arteriviruses have evolved multiple strategies to avoid elimination from the host. These tactics range from the prevention of detection to inhibition of antiviral responses mounted by the host immune system. All these activities involve virus-host interactions at different levels ( Figure 2) . The modulation of SARS-CoV pathogenesis, for example, was reported to be independent of all three types (Types I, II and III) of interferon signaling mechanisms, with SARS-CoV ORF 3b, ORF 6, and nucleocapsid proteins identified to interfere with interferon signaling through various mechanisms [94] . However, STAT1 has been shown by the same group to be crucial in activating innate immune signaling pathways during SARS-CoV infection, with a secondary role in the prevention of uncontrolled cell reproduction [95] . In contrast, PRRSV appears to be receptive to both IFN-α (porcine IFN-α, or Ad5-pIFN-α) and -β (recombinant swine beta interferon, or swIFN-beta) dose-dependent treatment [96, 97] ; however, pigs infected with PRRSV do not evoke significant IFN responses, with little or no IFN-α and IFN-β production [98, 99] . Upstream signaling pathways that may potentially lead to the inhibition of IRF3 activation include the interference of PRRSV with RIG-I signaling events through inactivation of the RIG-I downstream signaling adaptor, MAVS (mitochondrial antiviral signaling protein) [100] . Recent studies have also suggested PRRSV infection inhibits type I IFN production and signaling processes through the impairment of STAT1/STAT2 nuclear translocation [101] . One of the most indispensible adaptor proteins in the RLH signaling pathway is MAVS. Otherwise known as virus-induced signaling adaptor (VISA), interferon  (IFN) promoter stimulator-1 (IPS-1) or CARD-adaptor inducing IFN-β (Cardif), it was discovered independently by four different research groups in 2005 [102] [103] [104] [105] , and contains an N-terminal CARD-like domain, which interacts with the CARD domains of RIG-I and MDA5, and a C-terminal transmembrane (TM) region that lodges the protein in the mitochondrial membrane, thus pioneering the connection between mitochondria and innate immunity. A deficiency in, or the cleavage of, MAVS from the mitochondrial membrane results in an arrested antiviral immune response, highlighting the pivotal role MAVS plays in mitochondriamediated innate antiviral immunity [106] . MAVS can also induce apoptosis independently of Type I IFN activation, with SARS-CoV non-structural protein 15 identified as an inhibitor of MAVS-induced apoptosis to escape host antiviral immune responses [107] . Coronaviruses have devised a number of cell type-specific strategies to inhibit type I IFN responses. One such strategy that these viruses have adopted is to avoid detection of its newly synthesized mRNAs in the cytoplasm through the encoding of a 2'-O-methylase (non-structural protein nsp16) that creates a 5'-cap structure analogous to the cellular mRNAs on their mRNAs, thereby escaping detection by Mda5 [108, 109] . More importantly, this also highlights the significance of mRNA cap alterations, as such 2'-O-methylation, as pertinent molecular signals for the discernment between self and non-self mRNA [108] . However, during the course of viral transcription and replication, uncapped double-stranded RNA intermediates are generated and these may serve as ligands for the RIG-I and/or Mda5. Mouse hepatitis virus (MHV) infection was first reported to activate type I IFN responses in various cell types such as macrophages and microglia in the brains of infected animals, and the virus has also been shown in the same report to be recognized specifically by MDA5 in mouse macrophages [110] . In other reports, both RIG-I and Mda5 have been implicated in the detection of mouse hepatitis virus (MHV) infection, particularly in mouse oligodendrocytes [111] , although conflicting evidence has been reported as well, especially since it is not understood how cellular helicases could bind viral replicative/transcriptive intermediates when the latter should have been isolated by the double membrane vesicles [112] . MHV infection can also delay IFNβ-activated ISG induction; however this phenomenon is limited to certain cell types and is observed only when the infection occurs before IFNβ exposure [113] . As such, the general sensing of coronaviruses appears to be cell-type specific, with Type I IFNs derived from plasmacytoid dendritic cells (pDCs), conventional DCs and macrophages being particularly essential in curbing the pathogenesis of mouse coronavirus infection [114] . Detection by the PRRs would activate signaling cascades leading to the production of type I IFNs, resulting in the establishment of an antiviral state. Thus, coronaviruses do not just avoid detection by the host immune system, which appears to be the main strategy of ensuring successful replication [115] ; some viruses also encode proteins that function to disrupt the downstream signaling cascades at various points, preventing the establishment of an effective antiviral state when detection of the viral PAMPs has occurred. Indeed, the N protein has also been shown to interfere with the 2', 5'-oligoadenylate synthetase/RNaseL (2'-5'OAS) activation that occurs downstream of IFN induction and which leads to the inhibition of global translation shutdown [116] . This activity is in addition to its suppression of IFNβ induction through the binding of viral RNAs that prevents their detection [117] . In addition to the processing of polyproteins, the papain-like proteinase 2 (PLP2) domain of nsp3 has been shown to possess de-ubiquitinating activity [118] [119] [120] . PLP2 of nsp3 de-ubiquitinates TANK-binding kinase 1 (TBK1), the activating kinase for IRF3 and IRF3 itself, and sequesters the hypo-phosphorylated TBK1-IRF3 complex in the cytoplasm as well. This prevents IRF3 nuclear translocation [121, 122] , thereby inhibiting the transcription of type I interferons [123] . The activation of TLRs-3 and/or -7 as well as cytoplasmic helicases RIG-I and/or MDA-5 triggers signaling pathways resulting in the synthesis of type I IFNs, inflammatory cytokines and ISGs which acts in concert to establish an antiviral state. The activation of both 2'-5' OAS and PKR results in global degradation of cellular RNA and inhibition of translation, which may inhibit viral propagation. Coronaviruses encode many proteins (see yellow boxes) that target multiple steps in the innate immune response mounted by the host cells, ensuring its successful replication in the host. In vitro activation of chicken splenocytes and peripheral blood leukocytes with IBV has also resulted in an increase in chicken interferon gamma (chIFN-γ) production as a form of cell-mediated immune response [124] . This appears to be a polyclonal, non-specific stimulation as chIFN-γ production levels are also elevated in IBV-stimulated chicken splenocytes which lack prior exposure to IBV, when compared to control un-stimulated cells, as well as in cells exposed to inactivated IBV [124] . Such ineffectual host innate responses could lead to poor cellular responses, which may result in a delay in pathogen clearance and persistent viral infection in compromised pigs, which would present significant advantages to the virus for the subsequent release of viral progeny and spread. The onset of coronavirus-induced apoptosis is intricately linked with other host antiviral innate defenses. Infections with viruses often result in cell cycle arrests and the activation of unfolded protein response (UPR) due to ER stress, both of which may be accompanied by the parallel activation of apoptosis in the infected cells. During virus replication, newly translated viral proteins accumulate in the ER, which may also cause stress that leads to activation of the UPR. MHV S protein has been shown to activate three UPR transducers, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) [125] . In particular, PERK activation leads to the activation of p38 MAPK [126] , which stimulates virus replication [127, 128] . SARS-CoV, on the other hand, has been shown to attenuate the IRE1 signaling pathway, which generally led to the down-regulation of stress responses and UPR, and further decreased apoptotic signaling in infected cells as well, to the likely benefit of the virus for better viral progeny production and release [129] . Coronavirus-induced apoptosis during the late stages of infection is also partially regulated by the p38 MAPK signaling pathway, which in turn up-regulates production of pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, in infected host cells and mount an immune response against virus infection in these cells [130, 131] . However, while pro-inflammatory cytokines are up-regulated at the transcriptional level during coronavirus infection, there is minimal to moderate up-regulation at the translational level, leading to the hypothesis that the interaction between IBV spike (S) protein and host eukaryotic initiation factor 3 (eIF3) modulates host gene expression, especially genes involved in innate immunity that are activated during coronavirus infection [132] . This also agrees with the hypothesis that, notwithstanding the presence of virus-induced ER stress responses, a significant decline in host mRNA translation in infected cells impedes the expression of ER stress proteins despite elevated mRNA concentrations of the former [133] . Moreover, the negative modulation of p38 MAPK occurs through an up-regulation of the dual-specificity phosphatases 1 (DUSP1) feedback loop during IBV infection, which reduces cytokine production by dephosphorylation of phosphor-threonine and phosphor-tyrosine residues on activated p38 MAPKs [130] . PRRSV infection of porcine macrophages, too, appears to be under the control of the MAPK/ERK pathway, through various signaling pathways activated by the latter that regulate a variety of cellular processes [134] . In particular, chemical inhibition of the ERK pathway resulted in an attenuation of PRRSV infection during the early stages of infection post-virus attachment, during which a notable reduction in viral sub-genomic mRNA synthesis and translation, as well as in progeny virus release, was observed [134] . An increase in IL-10 production during the early stages of infection has also been observed in pigs infected with PRRSV. The up-regulation of IL-10, an important cytokine in the attenuation of innate and adaptive immune responses, is advantageous for PRRSV to sustain for a longer period of time in the host, and the PRRSV N protein has been suggested as the viral protein responsible for mediating IL-10 induction [135] . Likewise, the expression of pro-inflammatory mediators, IL-1, IL-6, IL-8, and TNF-α, are up-regulated during EAV infection of equine endothelial cells and macrophages, with virulent EAV strains activating sufficiently greater amounts of these cytokines, especially TNF-α, than avirulent EAV strains [136] . Moreover, the expression of pro-inflammatory cytokines, IFN-α, TNF-α, IL-1 and IL-6, is also up-regulated during porcine respiratory coronavirus and TGEV infection, and the differential modulation of which are postulated to function as crucial mediators of viral respiratory diseases in pigs [41] . In particular, early type I IFN production in coronavirus-infected pigs effectuated immunomodulatory responses, which suggests the potential ability of IFN inducers as an effective antiviral strategy to control the rate of coronavirus infections in swine populations [137] . RANTES (regulated upon activation, normal T cell expressed and secreted) is a known member of the pro-inflammatory CC chemokines family that function in the modulation of the migration of inflammatory cells such as monocytes and Natural Killer cells to sites of infection, particularly as a form of host antiviral immune response during virus infection [138] . Like coronaviruses, PRRSV infection also triggers the activation of pro-inflammatory cytokines and chemokines, including RANTES [139] , which may be fundamental in initiating the pathological conditions associated with the arterivirus. The activation of RANTES transcription also requires the participation of adaptor molecules from the TLR signaling pathway, such as MyD88, TRIF and TNF receptor-associated factor 6 (TRAF6) [139] . Interestingly, respiratory PRRSV/PRCV viral co-infections that commonly occurs in pigs, on the other hand, frequently leads to severely attenuated innate and adaptive immune responses in order to extend the pathogenicity of PRRSV-thus down-regulating innate immunity-and renders the host more vulnerable to subsequent infections by PRCV, or other respiratory viruses, that instead up-regulates innate immunity, possibly due to further, more severe damage to pulmonary cells and tissue and which may ultimately lead to a more critical form of pneumonia [140] . Other types of host innate defense against virus infection include processes such as autophagy, a vital physiological process in which cells degrade their own organelles in response to a severe lack of nutrients or other cellular stresses, so as to facilitate the removal of damaged cellular components and prevent the build-up of unwanted products in the cells [141] . Autophagy is also known to mount crucial innate immune responses against invading pathogens, with degradation of the latter through autophagy; on the other hand, autophagosomes may instead help promote virus infection by bringing together viral replicase proteins [142] . Multiple host-derived cytokines have emerged to play differential roles in regulating the onset of this essential mechanism as well. Of note are the T-helper cells (Th1) group of cytokines, including IFN-γ, TNF-α, IL-1, IL-2 and IL-6, which have the ability to induce autophagy, while Th2 cytokines such as IL-4, IL-10 and IL-13 have been shown to inhibit autophagy [143] . Recent studies have shown the ability of IBV to induce autophagy during infection independently of cellular stress or nutrient deprivation, and this can be made possible through the functions of IBV nsp 6, as well as in nsp6 orthologs of mammalian coronaviruses such as MHV and arteriviruses such as PRRSV, although a direct link between IBV infection and autophagosome formation could not be established [22] . As such, the induction of autophagy that has previously been reported during coronavirus and arterivirus replication may instead be an example of an innate defense tactic against infection to remove unwanted viral particles, while nsp6-as well as its respective orthologs-may alternatively modify adaptive immune strategies by targeting the breakdown of immunomodulatory proteins synthesized by the ER in autophagosomes [22] . Viruses may also exploit the host cell cycle to benefit their own replication [144] (Figure 3 ). Cell cycle regulation typically involves mechanisms critical to cell sustainability, such as the surveillance and correction of genetic damage and the impediment of unrestrained cell division. Cyclins and cyclin-dependent kinases (CDKs) are well-known regulatory proteins, and the activation of which will dictate a cell's progress through the cell cycle. Briefly, activated heterodimers consisting of both cyclins and CDKs as the regulatory and catalytic subunits, respectively, will alter the phosphorylation state of various target proteins for progression into the next stage of the cell cycle. CDK inhibitors include tumor suppressors such as the cip/kip (CDK interacting protein/Kinase inhibitory protein) and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) family of genes that thwart progression to the next stage of the cell cycle [145] . The inactivation of CDKs can also be a reversible process through the phosphorylation of essential residues Tyr 15 and Thr 14, both of which are located within the CDK ATP-binding loop [146] . Dual-specificity phosphatases of the Cdc25 (cell division cycle 25) family are responsible for the dephosphorylation of Tyr 15 and Thr 14 [147] . These Cdc25 proteins are, in turn, inactivated by Chk1/Chk2-mediated phosphorylation; this inactivation averts Cdk dephosphorylation and impedes its subsequent activation [148] . Chk1/Chk2 activation after DNA damage and/or DNA replication hindrance is dependent on the ataxia-telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) protein kinases in mammalian cells [149, 150] . IBV infection of cultured cells results in cell cycle arrest, at both S and G2/M phases, to boost viral replication and to enhance the production of viral proteins as well. This p53-independent growth inhibitory outcome is catalyzed by regulation of the expression of multiple cell cycle regulatory genes such as corresponding CDK complexes [50] and the down-regulation of down-regulation of G1 phase regulatory cyclins D1 and D2 [151] , as well as through systemic modulation of ATR-dependent cellular DNA damage response [152] . Specifically, the interaction of coronavirus nsp13 with the p125 subunit of DNA polymerase  was discovered to trigger DNA replication stress in cells during IBV infection, which eventually led to cell cycle arrest at the S phase [152] . It has also been observed that the N protein of several coronaviruses can localize in the nucleolus where it may perturb cell cycle activities of the host cell for the benefit of viral mRNA synthesis [153] [154] [155] [156] . IBV N, for example, appears to target CDK2, cyclins A and D1 for proteasomemediated degradation [50, 157] and cause the accumulation of hypophosphorylated retinoblastoma (pRB), resulting in the downregulation of CDK1, cyclins E and B1 [50] . The regulation of host protein synthesis, especially at the initiation stage, is often a regular viral objective for the extensive reduction of host protein translation so as to construct the most favorable condition for viral replication and progeny assembly. Protein kinase R (PKR) is a prevalent serine/threonine protein kinase that can be induced by interferon in its latent state, and whose activation is dependent by the presence of dsRNA products, is one such viral target. PKR plays an important role in the cellular anti-viral response pathway, and becomes activated via auto-phosphorylation upon binding to virus-derived dsRNA, which then subsequently leads to host translation inhibition through phosphorylation of the alpha subunit of eukaryotic initiation factor 2, eIF2alpha [158] . The dephosphorylation of eIF2alpha is established by cellular protein phosphatase-1 (PP1), which functions to regular various cellular processes through the physical interaction of its catalytic subunit (PP1c) with regulatory proteins such as GADD34/CHOP that is induced by DNA damage signals [159, 160] . In the case of IBV, for example, eIF2alpha phosphorylation was significantly suppressed in both human and animal cells during infection. The dephosphorylation of eIF2alpha during IBV infection appears to be induced by the up-regulation of GADD34 expression, which, together with the simultaneous attenuation of PKR auto-phosphorylation following IBV infection in these cells, serves as a viral regulatory tactic in boosting coronavirus replication while managing the delicate balance of de novo protein translation along with the identification of IBV nsp2 as a potential, albeit weak, mediator in the dose-dependent inhibition of PKR activation [161] . TGEV, too, suppresses both cellular RNA degradation and eIF2a phosphorylation during infection through an interaction between TGEV protein 7 and PP1 that regulates host antiviral responses and extends the period of viral progeny dispersal as well [53] . A subunit of the eukaryotic initiation factor 3, eIF3f, has also been discovered to modulate host translation inhibitory effects through physical interaction with coronavirus spike protein [132] . As this inhibition takes place during the late stages of the coronavirus replication cycle, translation of virus-induced transcripts are largely affected, especially pro-inflammatory cytokines and chemokines. This could account for the fact that while IL-6 mRNA expression is up-regulated during the early stages of coronavirus infection, insubstantial increase in IL-6 protein expression was observed [132] . The inhibition of host protein synthesis through the interaction between coronavirus spike protein and eIF3f may therefore have a significant impact on the modulation of coronavirus pathogenicity. Host proteins are also known to play a role in the virus life cycle, especially during viral RNA replication and transcription (Figure 4) . The most well studied host protein that interacts with the coronavirus genome is heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a nuclear protein, whose biological function is to regulate alternative splicing of cellular RNAs [162, 163] . The hnRNP A1 has been shown to bind to both negative-sense leader sequence and negative-sense intergenic (IG) sequence of MHV [164] . The formation of a RNP complex among hnRNP A1, negative-sense leader and IG sequences has also been demonstrated [165] . In addition to its ability to interact with the coronavirus RNA, hnRNP A1 was also found to interact and co-localize with N protein [166, 167] , an important player in coronavirus RNA synthesis [168, 169] . It has also been highlighted that hnRNP A1 may be required to recruit other cellular proteins to the replicase complex [170] . To ascertain the involvement of cellular factors in TGEV RNA synthesis, TGEV 3' and 5' genome ends were used as baits for RNA affinity protein purification [171] . Of the ten cellular proteins pulled down with either genome end, poly(A)-binding protein (PABP), hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) were confirmed to enhance TGEV infection through their respective interactions with the TGEV 3' end, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-originally employed as a control-was discovered, surprisingly, to have a diminishing effect on TGEV infection instead [171] . For arteriviruses, the common leader sequence of EAV sub-genomic viral RNAs, too, possesses the ability to interact several cellular proteins from the cytoplasmic fractions of Vero cells, likely for the modulation of EAV RNA synthesis [172] . Recent reports, based on a yeast-based three-hybrid system to identify RNA-RNA-binding regulatory protein interactions [173] , and using the 5'-UTR (untranslated region) of SARS coronavirus as bait, identified zinc finger CCHC-type and RNA-binding motif 1 (MADP1) as a potential cellular protein that interacts with SARS-CoV, and this was eventually established via an in vitro pull-down assay with the 5'-UTR of IBV [174] . MADP1 was also shown by the same authors to play a role in the early stages of the coronavirus replication cycle, with the RNA recognition motif in the N-terminal region of the protein interacting with stem loop 1 of the IBV 5'-UTR. Specifically, this protein translocates from the nucleus to the cytoplasm of the cell during IBV infection and co-localizes, in part, with viral replicase/transcriptase complexes (RTCs) in order to enhance viral replication and coronavirus RNA synthesis [174] . Interaction with viral RNA is not the only way by which cellular proteins can take part in viral RNA synthesis; protein-protein interactions with the viral replicase complex can modulate this process as well. This was confirmed in coronaviruses with a yeast two-hybrid screen that was performed using IBV nsp14 as a bait protein, which ultimately led to the discovery of DDX1, an ATP-dependent RNA helicase in the DExD/H helicase family, as an interacting partner that translocates from the nucleus to the cytoplasm and enhances IBV replication in cultured mammalian cells through subcellular colocalization with nsp14, an exonuclease, during infection [175] . Another yeast two-hybrid screen, with IBV membrane (M) protein as the bait, identified beta-actin as an interacting partner, leading to the suggestion that actin filaments may possess the ability to participate in virion assembly and budding during the coronavirus replication cycle [176] . This interaction may possibly lead to the incorporation of actin into the mature virion. In fact, recent proteomic analysis of purified IBV particles through two-dimensional gel electrophoresis and subsequent mass spectrometry have confirmed, among others, an abundance of actin within the virus particles, further cementing the hypothesis that cytoskeletal elements play crucial roles in IBV replication [177] . Retinoblastoma tumor suppressor proteins are also interacting partners of coronavirus nsp15, an endoribonuclease, which may result in alterations to the cell cycle that impact coronavirus infection and viral progeny release [178] . Similarly, with nsp1, the functional proteolytic product of PRRSV nsp1, as a bait protein, cellular poly(C)-binding proteins 1 and 2 (PCBP1 and PCBP2, respectively) have been identified interacting partners of the former, with specific functions in modulating PRRSV replication and RNA synthesis [179] . . Host proteins and the coronavirus life cycle. Virus particle attaches onto the host cell via cellular receptors on the surface and enters. Entry is followed by the uncoating of the ribonucleocapsid to expose the positive-sense genomic RNA which is translated by the host ribosomes to yield the viral replication complex. The viral replication complex continues with viral transcription and genome replication and, with the aid of host proteins such as hnRNPA1, yields a nested set of positive-sense sub-genomic sized mRNAs as well as the full-length virus genome. Sub-genomic sized mRNAs are translated by host ribosomes into viral structural (S, E, M, N) and accessory proteins. The N protein packages the positive-sense genomic RNA into a ribonucleocapsid and is assembled into the virus particles with the help of -actin. The newly formed virus particles undergo maturation when passing through the Golgi and exit the host cell via exocytosis. The relationship between a virus and its host is complex; the virus must evolve evasive strategies to avoid detection and immunological defense mechanisms from the host while the host must develop various lines of defense in order to combat viral invasion. As highlighted in this review, the diverse virus-host interactions established during animal coronavirus and arterivirus infections may have direct implications on viral replication itself, or they may also lead to the modification of numerous signaling pathways, such as cellular stress and/or host antiviral innate immune response, as a means to expedite viral replication and pathogenesis. The intricate network between a virus and its host is a complicated affair that involves many players-derived from both virus and host-which are pitted against one another in the battle for ascendancy; certain host processes are readily assimilated and manipulated by the virus in its bid to impede host antiviral responses while the host is itself armored with several lines of defense mechanisms and numerous antiviral factors to combat viral invasion to prevent its spread. While much progress has been made in the elucidation of the various regulatory pathways that, under the most propitious of conditions, will allow virus and host to co-exist in an uneasy truce, a clearer understanding of the complex interplay between the virus and its host could give new insights into the role of main players, such as those that govern the onset of apoptosis, or type I interferons and their regulators, and lead to the discovery of novel and/or universal targets for the therapeutic mediation against pathogenic infection.	What is the structure of a coronavirus?	5,314	large, non-segmented, positive sense and single stranded RNA animal viruses	1,119
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What method is developed in this study?	2,754	a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection.	840
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What is the model simplified to?	2,755	Reservoir-People (RP) transmission network model	1,216
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What is the estimate of R 0?	2,756	The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.5	1,439
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What is the conclusion of this study?	2,757	Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea.	1,716
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the focus of the study?	2,758	Huanan Seafood Wholesale Market (reservoir) to people,	3,803
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What were the model assumptions?	2,759	the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection	5,011
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What compartments were the bats divided into?	2,760	susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ).	5,425
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What compartments were the host animals divided into?	2,761	The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H )	5,942
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the SARS-COV-2 reservoir?	2,762	the seafood market	6,547
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What were the people divided into?	2,763	into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people.	7,197
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the mean incubation period?	2,764	5.2 days (95% confidence interval [CI]: 4.1-7.0)	9,998
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the mean delay from symptom onset to detection/hospitalization of a case?	2,765	5-day	10,193
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	How long after onset, the cases detected in Thailand and Japan were hospitalized?	2,766	from 3 to 7 days	10,323
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the duration from illness onset to first medical visit ?	2,767	a mean of 5.8 days (95% CI: 4.3-7.5)	10,518
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the assumption of transmissibility of asymptomatic infection?	2,768	0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza	10,958
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	As of January 17, how many people were tested for body temperature?	2,769	0.3 million people	11,379
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What is mobile population in Wuhan?	2,770	about 2.87 million	11,463
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the R0 of SARS?	2,771	2.7-3.4 or 2-4 in Hong Kong	15,034
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the value of R0 in other researches?	2,772	R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China	15,112
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What is the reported value of R0 for MERS?	2,773	0.8-1.3	15,455
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was R0 for the high transmissibility in South Korea?	2,774	2.5-7.2	15,674
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What is important for containing the transmission?	2,775	to decrease R 0	15,929
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What did this model show?	2,776	the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea.	17,978
2,592	A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.	What was the objective of the study?	2,777	provide a mathematical model for calculating the transmissibility of SARS-CoV-2	18,174
2,551	Potential Maternal and Infant Outcomes from (Wuhan) Coronavirus 2019-nCoV Infecting Pregnant Women: Lessons from SARS, MERS, and Other Human Coronavirus Infections https://doi.org/10.3390/v12020194 SHA: 779c1b5cb3afe3d50219aa2af791014a22eb355a Authors: Schwartz, David A.; Graham, Ashley L. Date: 2020 DOI: 10.3390/v12020194 License: cc-by Abstract: In early December 2019 a cluster of cases of pneumonia of unknown cause was identified in Wuhan, a city of 11 million persons in the People’s Republic of China. Further investigation revealed these cases to result from infection with a newly identified coronavirus, termed the 2019-nCoV. The infection moved rapidly through China, spread to Thailand and Japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents. Similar to such other coronaviruses as those causing the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), the new coronavirus was reported to spread via natural aerosols from human-to-human. In the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations. Unfortunately, there is limited experience with coronavirus infections during pregnancy, and it now appears certain that pregnant women have become infected during the present 2019-nCoV epidemic. In order to assess the potential of the Wuhan 2019-nCoV to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of SARS, MERS, and other coronavirus infections on pregnant women and their infants. Recommendations are also made for the consideration of pregnant women in the design, clinical trials, and implementation of future 2019-nCoV vaccines. Text: Coronaviruses are spherical, enveloped, and the largest of positive-strand RNA viruses. They have a wide host range, including birds, farm animals, pets, camels, and bats, in which they primarily cause respiratory and gastrointestinal disease. Belonging to the order Nidovirales, family Coronaviridae, and the subfamily Orthocoronaviridae there are four genera of coronaviruses-Alphacoronavirus, Betacoronavirus, Deltacorona virus, and Gammacoronavirus [1] [2] [3] [4] . In humans, they are a cause of mild illnesses including the common colds occurring in children and adults, and were believed to be of modest medical importance. However, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-can produce severe lower respiratory In the beginning of December 2019, a cluster of persons with a pneumonia of unknown cause was identified in Wuhan, the capital of Hubei Province and a large city of approximately 11 million persons located in the central region of the People's Republic of China [7, 8] . Between 8 and 18 December 2019 there were 7 cases of pneumonia identified whose clinical features resembled that of a viral pneumonia. The outbreak was initially believed to be linked to the Wuhan Huanan (South China) Seafood Wholesale Market. This market, termed a "wet" market, sells a variety of seafood, cuts of meat, and both live and dead animals in over one thousand stalls in constant close contact; however, whether this market was the origin of the outbreak remains unknown [9] . On 31 December 2019, the Chinese Center for Disease Control and Prevention (China CDC) sent a rapid response team to Hubei to work alongside health personnel from the provincial and Wuhan city health departments to conduct an epidemiologic investigation. As the disease was spreading through secondary and tertiary cases, the World Health Organization (WHO) China Country Office was informed on 31 December 2019 of the occurrence of these cases of pneumonia of unknown etiology. During the period from 31 December 2019 to 3 January 2020, 44 patients with pneumonia of unknown etiology were reported by the Chinese authorities to the WHO. On 7 January 2020 investigators in China identified the etiological agent of the epidemic as a previously unknown coronavirus, and it was given the designation 2019-nCoV (for 2019 novel coronavirus) [8] . Analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-nCoV infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old. The most common symptoms at the beginning of their illness included fever (40, 98%) , cough (31, 76%) , and fatigue or myalgia (18, 44%) , sputum production (11, 28%) , and headache (3, 8%) [10] . Among these 41 initial cases of 2019-nCoV infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ARDS), 13 (32%) required intensive care and 6 (15%) died. During the first weeks of January the infection spread rapidly through China and extended to adjacent countries where cases began to appear-13 January in Thailand, 15 January in Japan, 20 January in the Republic of Korea, and Taiwan and the United States on 21 January [11] . Infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of Wuhan. The new coronavirus continued to spread throughout multiple countries and continents, and by 9 February 2020 the WHO reported 37,251 confirmed cases in China that resulted in 812 deaths, surpassing the number of deaths that occurred during the 2002-2003 SARS epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . Viruses 2020, 12, 194 3 of 16 epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include premature rupture of membranes (PROM) and preterm labor (PTL), intrauterine fetal demise (IUFD), intrauterine growth restriction (IUGR), and neonatal death [14] [15] [16] . The SARS epidemic began quietly at the turn of the 21st century. In November 2002, a cook in Guangdong Province, China, died from an unidentified illness. He had worked at a restaurant in which meat from wild animals was served. On 27 November 2002 Chinese-language media and internet reports were picked up by Canada's Global Public Health Intelligence Network (GPHIN) that indicated a flu-like illness was occurring in China [25, 26] . Unfortunately, the reports were not translated, and China failed to report the occurrence of this illness to the World Health Organization (WHO) until February 2003. The disease spread to other countries where it primarily infected healthcare workers. One of these was Dr. Carlo Urbani, a WHO physician investigating a patient with the new disease in Hanoi. He recognized that the pneumonia was probably caused by a new, highly infectious agent, and rapidly notified the WHO. He contracted the SARS-CoV while there, became febrile and later died after traveling to Thailand to attend a conference. On 12 March 2003, WHO issued a global alert regarding the disease that was occurring primarily among health care workers in Hanoi, Vietnam and Hong Kong. The disease continued to spread, and by 31 July 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland China and Hong Kong. Approximately 30% of infections occurred in healthcare workers. By the termination of the epidemic the global CFR was 11% [27] . Although there were relatively few documented cases of SARS occurring during pregnancy, several case reports and small clinical studies have described the clinical effects in pregnant women and their infants. In reviewing these reports describing pregnant women with SARS in China it is possible, and perhaps even probable, that some of the same patients were included in more than one publication. However, even if this is the case, there is no doubt that SARS coronavirus infection was found to be associated with severe maternal illness, maternal death, and spontaneous abortion [19, [28] [29] [30] [31] . Martha Anker, an expert in statistics formerly with the WHO and the University of Massachusetts, estimated that more than 100 cases of SARS-CoV infection occurred in pregnant women, which warrants closer inspection [27] . The clinical outcomes among pregnant women with SARS in Hong Kong were worse than those occurring in infected women who were not pregnant [32] . Wong et al. [29] evaluated the obstetrical outcomes from a cohort of pregnant women who developed SARS in Hong Kong during the period of 1 February to 31 July 2003. Four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by SARS-related acute respiratory distress. Among the 5 women who presented after 24 weeks gestation, 4 had preterm deliveries (80%). A case-control study to determine the effects of SARS on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the Princess Margaret Hospital in Hong Kong [27, 33] . There were 3 deaths among the pregnant women with SARS (maternal mortality rate of 30%) and no deaths in the non-pregnant group of infected women (P = 0.006). Renal failure (P = 0.006) and disseminated intravascular coagulopathy (P = 0.006) developed more frequently in pregnant SARS patients when compared with the non-pregnant SARS group. Six pregnant women with SARS required admission to the intensive care unit (ICU) (60%) and 4 required endotracheal intubation (40%), compared with a 12.5% intubation rate (P = 0.065) and 17.5% ICU admission rate (P = 0.012) in the non-pregnant group. Maxwell et al. [32] reported 7 pregnant women infected with SARS-CoV who were followed at a designated SARS unit-2 of the 7 died (CFR of 28%), and 4 (57%) required ICU hospitalization and mechanical ventilation. In contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with SARS-CoV. Two women with SARS recovered and maintained their pregnancy but had infants with IUGR. Among the live newborn infants, none had clinical or laboratory evidence for SARS-CoV infection. The new mothers who had developed SARS were advised not to breastfeed to prevent possible vertical transmission of the virus. Zhang et al. [34] described SARS-CoV infections in 5 primagravidas from Guangzhou, China at the height of the SARS epidemic. Two of the mothers became infected in the 2nd trimester, and 3 developed infection in the 3rd trimester. Two of the pregnant women had hospital-acquired SARS infections, and the other 3 were community-acquired. All 5 pregnant women had fever and abnormal chest radiographs; 4 had cough; 4 developed hypoalbuminemia; 3 had elevated alanine aminotransferase levels (ALT), 3 had chills or rigor, 2 had decreased lymphocytes, and 2 had decreased platelets. One pregnant woman required intensive care, but all recovered and there were no maternal deaths. The 5 infants were clinically evaluated, and none had evidence of SARS. Two pregnant women with SARS were reported from the United States. In a detailed case report, Robertson et al. [35] described a 36-year-old pregnant woman with an intermittent cough of approximately 10 days duration and no fever. While travelling in Hong Kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with SARS-CoV. At 19 weeks gestation she developed fever, anorexia, headache, increasing cough, weakness, and shortness of breath. Upon returning to the United States she was hospitalized with pneumonia. Obstetrical ultrasounds revealed a low-lying placenta (placenta previa) but were otherwise normal. Following her discharge home and clinical recovery, she was found to have antibodies to SARS-CoV. She underwent cesarean section at 38 weeks gestation because of the placenta previa and a healthy baby girl was delivered [35, 36] . The placenta was interpreted as being normal. At 130 days post-maternal illness, maternal serum and whole blood, swabs from maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk were collected for analysis-no viral RNA was detected in specimens tested by reverse transcriptase polymerase chain reaction (RT-PCR). Antibodies to SARS-CoV were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (EIA) and indirect immunofluorescence assay. No clinical specimens (except for cord blood) were available for testing from the infant. The second case in the USA occurred in a 38-year-old woman who had travelled to Hong Kong at 7 weeks gestation where she was exposed to SARS-CoV in the same hotel as the aforementioned American woman [37] . Following her return to the United States, her husband developed the clinical onset of SARS, and 6 days later she became ill with fever, myalgia, chills, headache, coryza, and a productive cough with shortness of breath and wheezing. Following her hospitalization for SARS she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to SARS-CoV by enzyme immunoassay and immunofluorescent assays. Her pregnancy continued and was unremarkable except for developing elevated glucose levels. A cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy. At the time of delivery, the mother's serum samples were positive for antibodies to SARS-CoV, but samples taken of umbilical cord blood and placenta were negative. Breast milk sampled 12 and 30 days after delivery were also negative for SARS-CoV antibodies. Specimens evaluated from maternal blood, stool, and nasopharynx samples, as well as umbilical cord blood of the infant, were all negative for coronavirus RNA by RT-PCR. Neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral RNA. From Canada, Yudin et al. [38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates. She had acquired SARS from contact with an infected family member. Following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection. She had a normal labor and delivery and her newborn girl had no evidence of infection. In a study of 5 liveborn neonates who were delivered to women infected with SARS-CoV during the Hong Kong epidemic, results from multiple tests-including serial RT-PCR assays, viral culture, and paired neonatal serological titers-were negative for SARS-CoV [39] . None of the 5 neonates developed any clinical signs or symptoms of respiratory infection or compromise. Fortunately, there were no cases of vertical transmission identified among pregnant women infected with SARS-CoV during the 2002-2003 Asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of SARS have occurred. In the only reported study of the placental pathology of mothers with SARS, Ng et al. [41] reported the findings from 7 pregnant women infected with SARS-CoV. In the case of 2 women who were convalescing from SARS-CoV infection during the 1st trimester of pregnancy, the placentas were found to be normal. Three placentas were delivered from pregnancies in which the mothers had acute SARS-CoV infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta. In the placentas of 2 women who were convalescing from SARS-CoV infection in the 3rd trimester of pregnancy the placentas were highly abnormal. They showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion. These 2 pregnancies also were complicated by oligohydramnios and had poor obstetrical outcomes-both infants had developed IUGR. It is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas. Similar to other coronavirus infections, SARS-CoV is easily spread from person-to-person via respiratory droplets and secretions as well as through nosocomial contacts [42, 43] . In addition to transmission of SARS-CoV through natural aerosols from infected patients, it was found that in Hong Kong the SARS-CoV could also be transmitted by mechanical aerosols [44] . Environmental factors had an important role when it was discovered that during the Amoy Gardens housing estate outbreak as many as two-thirds of infected persons had diarrhea, SARS-CoV was excreted in their stools, and that aerosols arising from the flushing of toilets could transmit the virus [44] . Healthcare facilities were also an important source of new SARS infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection. In order to address the safety issues for the obstetrical management and delivery of pregnant women with SARS, guidelines were prepared by the Canadian Task Force on Preventive Health Care and the Society of Obstetricians and Gynaecologists of Canada [45] . These recommendations include: 1. "All hospitals should have infection control systems in place to ensure that alerts regarding changes in exposure risk factors for SARS or other potentially serious communicable diseases are conveyed promptly to clinical units, including the labour and delivery unit. At times of SARS outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for SARS. Upon arrival in the labour triage unit, pregnant patients with suspected and probable SARS should be placed in a negative pressure isolation room with at least 6 air exchanges per hour. All labour and delivery units caring for suspected and probable SARS should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation. If possible, labour and delivery (including operative delivery or Caesarean section) should be managed in a designated negative pressure isolation room, by designated personnel with specialized infection control preparation and protective gear. 5. Either regional or general anaesthesia may be appropriate for delivery of patients with SARS. Neonates of mothers with SARS should be isolated in a designated unit until the infant has been well for 10 days, or until the mother's period of isolation is complete. The mother should not breastfeed during this period. 7. A multidisciplinary team, consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anaesthesiologists, should be identified in each unit and be responsible for the unit organization and implementation of SARS management protocols. 8. Staff caring for pregnant SARS patients should not care for other pregnant patients. Staff caring for pregnant SARS patients should be actively monitored for fever and other symptoms of SARS. Such individuals should not work in the presence of any SARS symptoms within 10 days of exposure to a SARS patient. 9. All health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent spread of the SARS virus. 10. Regional health authorities in conjunction with hospital staff should consider designating specific facilities or health care units, including primary, secondary, or tertiary health care centers, to care for patients with SARS or similar illnesses." Middle East respiratory syndrome (MERS) was first reported in September 2012 in Saudi Arabia, following isolation of MERS-CoV from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] . In the 8 years since then, there have been more than 2494 confirmed cases of MERS resulting in upwards of 858 deaths globally [46] . While 27 countries have reported cases of MERS, approximately 80% of confirmed cases originated in Saudi Arabia [47] . To date, all known cases of MERS can be linked to travel or residence in countries along the Arabian Peninsula-that is, Bahrain; Iraq; Iran; Israel, the West Bank, and Gaza; Jordan; Kuwait; Lebanon; Oman; Qatar, Saudi Arabia; Syria; the United Arab Emirates (UAE); and Yemen [48] . The largest documented outbreak outside of this region occurred in 2015 in the Republic of Korea, in which 186 infections occurred, resulting in 38 deaths [49] . The index case in this outbreak reportedly returned from the Arabian Peninsula just prior to onset of illness [50] . MERS-CoV is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] . Nosocomial outbreaks in health care settings-the result of poor infection control and prevention-are widely recognized as the hallmark of MERS [1] . Superspreading events have been recorded in healthcare settings in Jordan, Al Hasa, Jeddah, Abu Dhabi and South Korea [47, [52] [53] [54] [55] . Like other coronaviruses, MERS-CoV can be spread through person-to-person contact, likely via infected respiratory secretions [48] . Transmission dynamics, however, are otherwise poorly understood [1] . Bats are believed to be the natural reservoir of MERS-CoV, and dromedary camels can have the virus and have been suggested as possible intermediary hosts as well as a source of infection to humans [2, 56, 57] . There are no clinical or serological reports of perinatal transmission of MERS, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (RSV) [58] . Researchers have not yet discovered ongoing transmission of MERS-CoV within communities outside of health care settings. The clinical presentation of MERS varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ARDS), septic shock, and multiple organ failure, often resulting in death. Most patients with MERS develop severe acute respiratory illness accompanied by fever, cough, and shortness of breath [50] . Progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] . MERS progresses much more rapidly to respiratory failure and has a higher case fatality rate than SARS [1] . Unlike SARS, however, infection with MERS-CoV is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] . The overall CFR of MERS is approximately 34.4% [46] . Most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk. As a result of the immunological changes that occur during pregnancy, women who are pregnant are included in this high-risk group. Pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of MERS-CoV infection; however, little is known of the pathophysiology of this infection during pregnancy. Limited data exists on the prevalence and clinical features of MERS during pregnancy, birth, and the postnatal period. It is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the MERS-CoV and the severity of clinical illness [60] . Pregnant women infected with SARS-CoV, a related coronavirus, appear to have increased morbidity and mortality when compared to non-pregnant women, suggesting that MERS-CoV could also lead to severe clinical outcomes in pregnancy. To date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes. Between November 2012 and February 2016, there were 1308 cases of MERS reported by the Saudi Arabia Ministry of Health (MoH). Of these, 5 patients were pregnant, according to a retrospective study by Assiri et al. [47] , and all resulted in adverse outcomes. Patient ages ranged from 27 to 34 years, with occurrence of exposure in either the 2nd or 3rd trimester. All 5 cases received intensive care. Two women died and there were 2 cases of perinatal death-1 stillbirth and 1 neonatal death shortly after emergency cesarean section. These instances of severe maternal and perinatal outcomes are consistent with other reports of MERS-CoV infection in pregnant women, as well as outcomes associated with SARS-CoV infection. The authors of the retrospectives study concede that unreported cases of MERS in pregnancy are likely due to lack of routine pregnancy testing [47] . They conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for MERS-CoV, to contribute to overall understanding of pathogenesis and epidemiological risk. Additionally, 2 of the 5 patients were healthcare workers, which corresponds with existing knowledge of higher risk of exposure to MERS-CoV in healthcare settings. In a separate case report of MERS occurring in pregnancy, Alserehi et al. [58] described a 33-year-old critical care nurse who became infected during the 3rd trimester in the midst of a large hospital outbreak. In the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus. Following an emergency cesarean section at 32 weeks gestation, she was transferred to the intensive care unit (ICU) and later recovered. The preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother. In contrast to other reported cases, this patient had a successful outcome, perhaps due to the timing of MERS-CoV exposure, her young age, the use of steroids, and differences in immune response. Alfaraj et al. [61] described 2 cases of maternal infection with MERS-CoV at the Prince Mohammed Bin Abdulaziz Hospital (PMAH) in Saudi Arabia. Maternal infection in both cases was confirmed by nasopharyngeal swab testing by RT-PCR. One patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions. The second patient, a 39-year-old at 24 weeks gestation, had several comorbidities, including end stage renal disease, hypertension, and hemodialysis. This woman presented to the hospital after contact with a MERS-CoV-infected person during an active outbreak. Both patients later tested negative for MERS-CoV and were subsequently discharged. The younger patient delivered a healthy, full-term infant. The status of the other delivery is unknown. Neither fetus was tested for MERS-CoV. According to Payne et al. [62] , epidemiologic investigation of the 2012 MERS outbreak in Zarqa, Jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to MERS-CoV. The mother experienced fever, fatigue, headache and cough, concurrently with vaginal bleeding and abdominal pain. On the 7th day of symptoms, she had a fetal death. The mother was confirmed to have antibody to MERS-CoV, and she self-reported having had unprotected contact with family members who later tested positive for the virus. This was the first documented occurrence of stillbirth during maternal infection with MERS-CoV. On 24 November 2013, a 32-year-old pregnant woman in the United Arab Emirates (UAE) developed ARDS following admission to the ICU after suspected community-acquired pneumonia advanced to respiratory failure and hypotension [60] . Later that day, her baby was delivered by caesarean section and subsequent Apgar scores were within healthy range. The next day, RT-PCR evaluation revealed that the mother was positive for MERS-CoV. Despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died. While the outcome for this mother was fatal, Malik et al. noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] . More research is needed to determine safety, efficacy, and dosage of these therapies in the general population but also in pregnant women. While few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] . Outside of the Middle East the only confirmed case of MERS in pregnancy occurred in 2015 in South Korea. Jeong et al. [49] reported that a 39-year-old patient was exposed during the 3rd trimester following contact with a patient having MERS. Despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation. Subsequent testing of the infant's blood did not detect any IgG, IgM, or IgA antibodies to MERS-CoV. The mean maternal age of the 11 confirmed maternal SARS cases described above was 33.2 years, with a mean gestational age of 26.3 weeks. The source of infection in 2 of the cases was attributed to contact with family members who tested positive for MERS-CoV, unknown in 3 cases, likely due to animal exposure in 1 case, and 6 were healthcare-associated (2 of these patients were healthcare workers). Six patients required intensive care and 3 died. Of those who died, 2 were exposed to MERS-CoV in the 3rd trimester, and 1 was exposed during the 2nd trimester. The infant death rate for all 11 cases was 27%. Fetal survival did not appear to correlate with the timing of maternal infection and gestational age; however, more data are needed to draw conclusions about this relationship. According to Alfaraj et al. [61] , the CFR for the 11 infected women-also 27%-was not statistically different from the overall CFR of MERS in the general population (35%) (P = 0.75). Only 1 case resulted in both maternal and fetal death. Similar to SARS in pregnancy, more research is needed to understand the pathogenesis and epidemiology of MERS in pregnancy including the relationship between the timing of maternal infection, gestational age of the fetus, the effects of comorbid factors, and the occurrence of adverse outcomes. Few studies documented the presence of MERS-CoV antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission. As such, future studies should involve the collection of samples from relevant specimens including amniotic fluid, placenta, and umbilical cord [49] . MERS prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] . Since 2013, the Saudi Arabia MoH has recommended that pregnant women postpone travel to Saudi Arabia for the Hajj and Umrah [47] . To further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended. Pregnant women who present with symptoms of pneumonia, influenza-like illness (ILI), or sepsis on the Arabian Peninsula may also benefit from MERS-CoV screening to expedite early diagnosis and improve disease management [60] . While multiple agents have been used to treat MERS, none have been tested in large clinical studies. Available data are limited to the use of combination therapies of interferon and other agents in case reports and case series [63] . A prospective or randomized study may prove difficult given the sporadic nature of MERS-CoV outbreaks. Due to a gap in research on the treatment of MERS in pregnancy, there are no therapeutic options currently recommended for pregnant women [58] . Therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects. For example, during the 2003 SARS outbreak, ribavirin was administered to pregnant women with severe cases of the disease, but ribavirin therapy has been documented to increase the risk of teratogenic effects in newborns [58] . The Alphacoronaviruses HCoV 229E and NL63, as well as the Betacoronaviruses HKU 1 and OC43, can infect humans and cause the common cold. In order to investigate the potential maternal-fetal transmission of human coronaviruses during pregnancy, Gagneur et al. [64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants. These specimens were evaluated for the presence of HCoV 229E, OC-43, NL63 and HKU 1 using RT-PCR methodology. Between the period from July 2003 to August 2005 the authors examined 159 mother-infant dyads. Human coronaviruses were identified in 12 samples (HCoV 229E: 11; HKU 1 : 1) from 7 mother-child pairs. In 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive. There were no signs of clinical infection in any of the 3 neonates that had positive gastric samples for human coronavirus. It is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans. There are clearly challenges to this endeavor-protective antibodies to coronaviruses are not long-lasting, tissue damage has been reported to occur as a result of exposure to SARS-CoV, development of animal models that closely resemble human infection are limited, and the extensive time and expense necessary to perform clinical trials in humans, to name a few [66] [67] [68] . It is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-nCoV. In examining the history of vaccine design, it is clear that the needs of pregnant women have rarely been prioritized in either the preclinical development or the clinical trial phases of production. Today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] . Excluding pregnant women and their infants from participation in vaccine development and implementation undermines ethical principles of justice-fairness, equity, and maximization of benefit-and potentially places their health at risk during outbreaks and other health emergencies [69] [70] [71] . On 23 January 2020 the Coalition for Epidemic Preparedness Innovations (CEPI) announced three programs to develop a vaccine against the novel Wuhan coronavirus. The Chief Executive Officer of CEPI, Richard Hatchett, said [72] : "Given the rapid global spread of the nCoV-2019 virus the world needs to act quickly and in unity to tackle this disease. Our intention with this work is to leverage our work on the MERS coronavirus and rapid response platforms to speed up vaccine development." The novel coronavirus is the first epidemic disease to emerge since the formation of CEPI in Davos in 2017. CEPI was created with the express intent to enable speedy research and development of vaccines against emerging pathogens. In May 2017, WHO released the Target Product Profile (TPP) for MERS-CoV vaccines, following the prioritization of MERS-CoV as one of eight priority pathogens for prevention of epidemics [73] . CEPI and partners aim to use existing platforms-that is, the existing "backbone" that can be adapted for use against new pathogens-that are currently in preclinical development for MERS-CoV vaccine candidates. Following the WHO declaration on 30 January that the current 2019-nCoV outbreak is a public health emergency of international concern (PHEIC), global health organizations and researchers will be further mobilized-bolstered by new mechanisms for action and greater resources-to stop the spread of disease. A critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development? As of the PHEIC declaration, 12 groups have announced that they are developing new vaccines against 2019-nCoV and seven others announced initiatives to develop new therapies [74] . Safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind. To date, very few clinical trials for vaccines have proactively included pregnant women [75] , and the exclusion of pregnant and lactating women from receiving the rVSV-ZEBOV vaccine through 3 Ebola virus epidemics serves as a recent example [69] [70] [71] . Given the potential severity in pregnancy, as demonstrated by this review of maternal infections of SARS and MERS, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses. On 5 February 2020 it was reported by multiple media outlets that a newborn infant delivered during the epidemic in Wuhan had tested positive for 2019-nCoV at the Wuhan Children's Hospital in Hubei Province 30 hours following its birth. According to the official Xinhua news agency, the infant was delivered on 2 February to a mother who had tested positive for the virus. Reports have stated that the infant had stable vital signs, no fever or cough, but had shortness of breath together with abnormal chest radiographs and abnormalities of liver function [76] [77] [78] . Dr. Zeng Lingkong, Chief Physician at the Neonatal Medicine Department of the hospital, said [78] , "This reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission" The hospital also provided information about a previous case of a baby that had been delivered on 13 January 2020. Following its birth, the infant's nanny was diagnosed with 2019-nCoV, and the mother was diagnosed days later [76] . On 29 January the baby began to develop symptoms. According to Dr. Zeng Lingkong [76] , "Whether it was the baby's nanny who passed the virus to the mother who passed it to the baby, we cannot be sure at the moment. But we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected" In considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission. In some cases, viral infection can be acquired when the infant passes through the birth canal during a vaginal delivery or through post-partum breast feeding, although these mechanisms would be highly unusual for a respiratory virus. Neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment. Based upon past experience with pregnant women who developed MERS and SARS, and realizing that the numbers are limited, there has never been confirmed intrauterine coronavirus transmission from mother to fetus. Discussing the most recent baby to be diagnosed with the 2019-nCoV infection, Dr. Stephen Morse, an epidemiologist at the Mailman School of Public Health at Columbia University stated [77] , "It's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat," "It's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing." And according to Dr. Paul Hunter, Professor of Medicine at the University of East Anglia [79] , "As far as I am aware there is currently no evidence that the novel coronavirus can be transmitted in the womb. When a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth." There is limited knowledge regarding coronavirus infections that occur during pregnancy-what is known has, for the most part, been the result of epidemics resulting from two different diseases, SARS and MERS. These previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support. Both of these coronaviruses can result in maternal death in a small but significant number of cases, but the specific risk factors for a fatal outcome during pregnancy have not been clarified. Coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the ICU, spontaneous abortion and perinatal death. Unlike some viral infections, notably Ebola virus [70] and Zika virus [80] , the likelihood of intrauterine maternal-fetal transmission of coronaviruses is low-there have been no documented cases of vertical transmission occurring with either SARS or MERS. It remains to be seen during the current Wuhan 2019-nCoV epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables. However, pregnant women should be considered to be at high risk for developing severe infection during this current outbreak of 2019-nCoV. Additional clinical research on the treatment of SARS, MERS, and the new coronavirus 2019-nCoV is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy. This research will be critical in improving the care, and even saving the lives, of pregnant women in the current as well as future outbreaks.	What are coronaviruses?	2,199	s are spherical, enveloped, and the largest of positive-strand RNA v	1,930

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